Reverse oxidative phosphorylation?

Reverse oxidative phosphorylation?

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I noticed that all of the cellular energy production methods that I covered have a fixed ratio of ATP to NAD(P)H out. For example, in the combined process of glycolysis, pyruvate oxidization, and the Krebs cycle, 10 NADH, 2 FADH2, and 4 ATP are produced (based off of my high school's bio text book, if I'm adding that right). In this way, NADH and FADH2 can easily be exchanged for ATP through oxidative phosphorylation. That got me wondering what would happen if a cell was using so many electrons (from NADH and/or FADH2) that there is too much ATP. (Just as a hypothetical: It seems fairly unlikely to me)

I was looking for a way that oxidative phosphorylation could be reversed, but I didn't find much. I did find that anaerobic bacteria can oxidize nitrite in this way, but I couldn't find much evidence of an O2-forming version. Does such a thing exist?

Chemiosmotic Theory

The Royal Swedish Academy of Sciences decided to award the 1978 Nobel Prize in Chemistry to
Dr Peter Mitchell, Glynn Research Laboratories, Bodmin, Cornwall, UK, for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory.

Chemiosmotic Theory of Energy Transfer

Peter Mitchell was born in Mitcham, in the County of Surrey, England, on September 29, 1920. His parents, Christopher Gibbs Mitchell and Kate Beatrice Dorothy (née) Taplin, were very different from each other temperamentally. His mother was a shy and gentle person of very independent thought and action, with strong artistic perceptiveness. Being a rationalist and an atheist, she taught him that he must accept responsibility for his own destiny, and especially for his failings in life.That early influence may well have led him to adopt the religious atheistic personal philosophy to which he has adhered since the age of about fifteen. His father was a much more conventional person than his mother, and was awarded the O.B.E. for his success as a Civil Servant.

Peter Mitchell was educated at Queens College, Taunton, and at Jesus college, Cambridge. At Queens he benefited particularly from the influence of the Headmaster, C. L. Wiseman, who was an excellent mathematics teacher and an accomplished amateur musician. The result of the scholarship examination that he took to enter Jesus College Cambridge was so dismally bad that he was only admitted to the University at all on the strength of a personal letter written by C. L. Wiseman. He entered Jesus College just after the commencement of war with Germany in 1939. In Part I of the Natural Sciences Tripos he studied physics, chemistry, physiology, mathematics and biochemistry, and obtained a Class III result. In part II, he studied biochemistry, and obtained a II-I result for his Honours Degree.

He accepted a research post in the Department of Biochemistry, Cambridge, in 1942 at the invitation of J. F. Danielli. He was very fortunate to be Danielli’s only Ph.D. student at that time, and greatly enjoyed and benefited from Danielli’s friendly and unauthoritarian style of research supervision. Danielli introduced him to David Keilin, whom he came to love and respect more than any other scientist of his acquaintance.

He received the degree of Ph.D. in early 1951 for work on the mode of action of penicillin, and held the post of Demonstrator at the Department of Biochemistry, Cambridge, from 1950 to 1955. In 1955 he was invited by Professor Michael Swann to set up and direct a biochemical research unit, called the Chemical Biology Unit, in the Department of Zoology, Edinburgh University, where he was appointed to a Senior Lectureship in 1961, to a Readership in 1962, and where he remained until acute gastric ulcers led to his resignation after a period of leave in 1963.

From 1963 to 1965, he withdrew completely from scientific research, and acted as architect and master of works, directly supervising the restoration of an attractive Regency-fronted Mansion, known as Glynn House, in the beautiful wooded Glynn Valley, near Bodmin, Cornwall – adapting and furnishing a major part of it for use as a research labotatory. In this, he was lucky to receive the enthusiastic support of his fornler research colleague Jennifer Moyle. He and Jennifer Moyle founded a charitable company, known as Glynn Research Ltd., to promote fundamental biological research and finance the work of the Glynn Research Laboratories at Glynn House. The original endowment of about £250,000 was donated about equally by Peter Mitchell and his elder brother Christopher John Mitchell.

In 1965, Peter Mitchell and Jennifer Moyle, with the practical help of one technician, Roy Mitchell (unrelated to Peter Mitchell), and with the administrative help of their company secretary, embarked on the programme of research on chemiosmotic reactions and reaction systems for which the Glynn Research Institute has become known. Since its inception, the Glynn Research Institute has not had sufficient financial resources to employ more than three research workers, including the Research Director, on its permanent staff. He has continued to act as Director of Research at the Glynn Research Institute up to the present time. An acute lack of funds has recently led to the possibility that the Glynn Research Institute may have to close.

Mitchell studied the mitochondrion, the organelle that produces energy for the cell. ATP is made within the mitochondrion by adding a phosphate group to ADP in a process known as oxidative phosphorylation. Mitchell was able to determine how the different enzymes involved in the conversion of ADP to ATP are distributed within the membranes that partition the interior of the mitochondrion. He showed how these enzymes’ arrangement facilitates their use of hydrogen ions as an energy source in the conversion of ADP to ATP.

Peter Mitchell’s 1961 paper introducing the chemiosmotic hypothesis started a revolution which has echoed beyond bioenergetics to all biology, and shaped our understanding of the fundamental mechanisms of biological energy conservation, ion and metabolite transport, bacterial motility, organelle structure and biosynthesis, membrane structure and function, homeostasis, the evolution of the eukaryote cell, and indeed every aspect of life in which these processes play a role. The Nobel Prize for Chemistry in 1978, awarded to Peter Mitchell as the sole recipient, recognized his predominant contribution towards establishing the validity of the chemiosmotic hypothesis, and ipso facto, the long struggle to convince an initially hostile establishment.


Mitchell’s research has been carried out within an area of biochemistry often referred to in recent years as ‘bioenergetics’, which is the study of those chemical processes responsible for the energy supply of living cells. Life processes, as all events that involve work, require energy, and it is quite natural that such activities as muscle contraction, nerve conduction, active transport, growth, reproduction, as well as the synthesis of all the substances that are necessary for carrying out and regulating these activities, could not take place without an adequate supply of energy.

It is now well established that the cell is the smallest biological entity capable of handling energy. Common to all living cells is the ability, by means of suitable enzymes, to derive energy from their environment, to convert it into a biologically useful form, and to utilize it for driving various energy requiring processes. Cells of green plants as well as certain bacteria and algae can capture energy by means of chlorophyll directly from sunlight – the ultimate source of energy for all life on Earth – and utilize it, through photosynthesis, to convert carbon dioxide and water into organic compounds. Other cells, including those of all animals and many bacteria, are entirely dependent for their existence on organic compounds which they take up as nutrients from their environment. Through a process called cell respiration, these compounds are oxidized by atmospheric oxygen to carbon dioxide and water.

During both photosynthesis and respiration, energy is conserved in a compound called adenosine triphosphate, abbreviated as ATP. When ATP is split into adenosine diphosphate (ADP) and inorganic phosphate (Pi), a relatively large amount of energy is liberated, which can be utilized, in the presence of specific enzymes, to drive various energy-requiring processes. Thus, ATP may be regarded as the universal ‘energy currency’ of living cells. The processes by which ATP is formed from ADP and Pi during photosynthesis and respiration are usually called ‘photophosphorylation’ and ‘oxidative phosphorylation’, respectively. The two processes have several features in common, both in their enzyme composition – both involve an interaction between oxidizing (electron-transferring) and phosphorylating enzymes – and in their association with cellular membranes. In higher cells, photophosphorylation and oxidative phosphorylation occur in specific membrane-enclosed organelles, chloroplasts and mitochondria, respectively in bacteria, both these processes are associated with the cell membrane.

The above concepts had been broadly outlined by about the beginning of the 1960s, but the exact mechanisms by which electron transfer is coupled to ATP synthesis in oxidative phosphorylation and in photophosphorylation remained unknown. Many hypotheses were formulated, especially with regard to the mechanism of oxidative phosphorylation most of these postulated a direct chemical interaction between oxidizing and phosphorylating enzymes. Despite intensive research in many laboratories, however, no experimental evidence could be obtained for any of these hypotheses. At this stage, in 1961, Mitchell proposed an alternative mechanism for the coupling of electron transfer to ATP synthesis, based on an indirect interaction between oxidizing and phosphorylating enzymes. He suggested that the flow of electrons through the enzymes of the respiratory or photosynthetic electron-transfer chains drives positively charged hydrogen ions, or protons, across the membranes of mitochondria, chloroplasts and bacterial cells. As a result, an electrochemical proton gradient is created across the membrane. The gradient consists of two components: a difference in hydrogen ion concentration, or pH, and a difference in electric potential the two together form what Mitchell calls the ‘protonmotive force’. The synthesis of ATP is driven by a reverse flow of protons down the gradient. Mitchell’s proposal has been called the ‘chemiosmotic theory’.

This theory was first received with scepticism but, over the past 15 years, work in both Mitchell’s and many other laboratories have shown that the basic postulates of his theory are correct. Even though important details of the underlying molecular mechanisms are still unclear, the chemiosmotic theory is now generally accepted as a fundamental principle in bioenergetics. This theory provides a rational basis for future work on the detailed mechanisms of oxidative phosphorylation and photophosphorylation. In addition, this concept of biological power transmission by protonmotive force (or ‘proticity’, as Mitchell has recently began to call it in an analogy with electricity) has already been shown to be applicable to other energy-requiring cellular processes. These include the uptake of nutrients by bacterial cells, cellular and intracellular transport of ions and metabolites, biological heat production, bacterial motion, etc. In addition, the chloroplasts of plants, which harvest the light-energy of the sun, and the mitochondria of animal cells, which are the main converters of energy from respiration, are remarkably like miniaturized solar- and fuel-cell systems. Mitchell’s discoveries are therefore both interesting and potentially valuable, not only for the understanding of biological energy-transfer systems but also in relation to the technology of energy conversion.

Oxidative phosphorylation inhibition induces anticancerous changes in therapy-resistant–acute myeloid leukemia patient cells

Correspondence Aida Vitkevičienė, Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio av. 7, LT-10257 Vilnius, Lithuania.

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Centre of Hematology, Oncology and Transfusion Medicine, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Proteomics Centre, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Proteomics Centre, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Centre of Hematology, Oncology and Transfusion Medicine, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Correspondence Aida Vitkevičienė, Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio av. 7, LT-10257 Vilnius, Lithuania.

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Centre of Hematology, Oncology and Transfusion Medicine, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Proteomics Centre, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Proteomics Centre, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Centre of Hematology, Oncology and Transfusion Medicine, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania


Treatment of acute myeloid leukemia (AML) is still a challenge because of common relapses or resistance to treatment. Therefore, the development of new therapeutic approaches is necessary. Various studies have shown that certain cancers, including some chemoresistant AML subsets, have upregulated oxidative phosphorylation. In this study, we aimed to assess treatment-resistant AML patients’ cell modulation using oxidative phosphorylation inhibitors metformin and atovaquone alone and in various combinations with cytosine analog cytarabine and apoptosis inducer venetoclax. Metabolic activity analysis using Agilent Seahorse XF Extracellular Flux Analyzer revealed that peripheral blood mononuclear cells’ metabolic state was different among treatment-resistant AML patients. We demonstrated that metformin decreased therapy-resistant–AML cell oxidative phosphorylation ex vivo, cotreatment with cytarabine and venetoclax slightly increased the effect. However, treatment with atovaquone did not have a marked effect in our experiment. Cell treatment had a slight effect on cell proliferation inhibition combination of metformin, cytarabine, and venetoclax had the strongest effect. Moreover, a slightly higher effect on cell proliferation and cell cycle regulation was demonstrated in the cells with higher initial oxidative phosphorylation rate as demonstrated by gene expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Proteomic analysis by liquid chromatography–mass spectrometry demonstrated that chemoresistant AML cell treatment with metformin modulated metabolic pathways, while metformin combination with cytarabine and venetoclax boosted the effect. We suggest that oxidative phosphorylation inhibition is effective but not sufficient for chemoresistant AML treatment. Indeed, it causes anticancerous changes that might have an important additive role in combinatory treatment.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


Thermodynamic feasibility and maximum DHB yield

The proposed pathway proceeds through the activation of the malate β-carboxylate group by phosphorylation followed by two successive rounds of reduction to yield DHB (Fig. 1). The negative standard Gibbs free energy for the pathway (Supplementary Note 1 Supplementary Table 1) attests to its thermodynamic viability. Stoichiometric analysis of the metabolic network in E. coli shows that DHB can be produced from glucose with a theoretical maximum yield of 1.5 mol mol −1 (Supplementary Note 2 Supplementary Fig. 1). Given that DHB can be converted into the methionine-analogue HMTB without carbon loss, the production of methionine via DHB increases the theoretical yield by ∼ 100% compared to the conventional one-step biosynthesis of methionine from glucose and sulfate, and by ∼ 30% when compared to the biosynthesis of methionine from glucose and methanethiol 10 .

The synthetic 2,4-dihydroxybutyrate (DHB) pathway is inspired by the natural homoserine pathway.

Given the structural similarity of DHB and homoserine pathway intermediates, we identified three homoserine pathway enzymes as prototype candidates for screening and engineering of enzyme activity in the DHB pathway. We chose AK, encoded by lysC 17 , and ASD, encoded by asd 18 , from E. coli, and HSD, encoded by HOM6 (ref. 19), from Saccharomyces cerevisiae since the HSD enzymes in E. coli are bifunctional enzymes with an associated AK activity 20,21 . We found that Ec-Asd had only trace activity on the synthetic substrate (kcat=0.13 s −1 on malyl-P versus 36 s −1 on aspartyl-4-phosphate (aspartyl-P), whereas Ec-LysC and Sc-Hom6 had no detectable activity on malate and malate semialdehyde, respectively (Supplementary Table 2 Supplementary Note 3). These findings indicated that the feasibility of the envisaged de novo synthetic pathway necessitated the engineering of all three enzyme activities.

Engineering of malate kinase activity

We first set out to engineer MK activity into aspartate kinase III from E. coli (Ec-LysC). Binding interactions of the ( L )-aspartate natural substrate in the enzyme active site are revealed in the 2.5 Å X-ray crystallographic structure of the dimeric wild-type LysC abortive ternary complex with ( L )-aspartate and the Mg-ADP reaction co-product in the R-state 22 . The amino-acid substrate is anchored in position via water-mediated interactions of the β-carboxylate group with the metal ion in Mg-ADP, and by respective salt bridge electrostatic interactions of the ( L )-aspartate α-amino and α-carboxylate groups with charged enzyme side-chain functional groups of Glu119 and Arg198 as shown in Supplementary Fig. 2. Productive binding of the ( L )-aspartate substrate is further supported by a network of van der Waals and hydrogen bonding interactions with other residues in the active site.

( L )-Malate is an isostere of ( L )-aspartate in which the positively charged α-amino group is replaced by an uncharged hydroxyl group. In common with other (succinate and malonate) structural analogues of ( L )-aspartate that do not contain an α-amino group, and which therefore cannot form a salt-bridge with the side-chain of Glu119, ( L )-malate has been reported to be a weak competitive inhibitor of Ec-LysC with a Ki of 53 mM (ref. 23). These observations suggest that ( L )-malate binds non-productively to the wild-type enzyme in the absence of additional binding energy provided by the salt-bridge interaction.

To investigate the impact of Glu119 substitution by other amino acids, we carried out saturation mutagenesis at this residue position and analysed the enzymatic activities of the resulting mutants. Trace but measurable enzymatic activity on ( L )-malate could be detected with the Ec-LysC E119Q and Ec-LysC E119N mutants, in which the Glu119 side-chain carboxylate group is replaced by an uncharged carbamoyl group (Supplementary Table 3). Significant increases of up to 200-fold in the kcat/Km value for ( L )-malate were obtained in E119G, E119A, E119S and E119C variants with shorter side-chains at residue position 119 (Fig. 2a Supplementary Table 3).

(a) Catalytic efficiency (kcat/Km) of wild-type aspartate kinase and the best malate kinase mutants on ( L )-aspartate and ( L )-malate. Kinetic data are from Supplementary Table 3. The results are the mean of at least two biological replicate experiments. Error bars correspond to the standard deviation of the mean. (b) Catalytic efficiencies (kcat/Km) of wild type LysC and the V115A E119S E434V Lys C triple mutant on ( L )-aspartate and ( L )-malate. Kinetic data are from Supplementary Table 3. (c) Active-site region in molecular model of complex of the E. coli (Ec-LysC) E119S:V115A double mutant with ( L )-malate and Mg-ADP. Carbon atoms in ADP and ( L )-malate are coloured in purple. The Mg 2+ ion is depicted as an ochre-coloured space filling sphere, and water molecules mediating binding interactions as yellow spheres. Enzyme residue positions in the combinatorial library for experimental screening of malate kinase activity (Supplementary Table 5) are highlighted with green (or cyan) coloured carbon atoms according to whether (or not) direct residue contact can be made with ( L )-malate in the model complex. Atoms are otherwise coloured according to element type: other carbon, grey nitrogen, blue oxygen, red and phosphorus, orange. Hydrogen bond interactions are shown as dashed-line vectors connecting donor and acceptor heavy atom positions. The model is overlaid on the X-ray structure of the R-state wild-type enzyme complex with ( L )-aspartate and Mg-ADP (PDB code 2j0w) from which it was derived as described in Methods. Diffuse molecular surface representations of the mutant and wild-type enzyme active-sites are respectively shown in green and grey.

To further improve the catalytic efficiency towards ( L )-malate, the construction of a small combinatorial mutant library was undertaken. Analysis of binding interactions of Ec-LysC in the experimental complex with ( L )-aspartate enabled the selection of eight amino-acid residue positions including Glu 119, highlighted in Supplementary Fig. 2. Of these eight positions, five make direct contact with the ( L )-aspartate substrate (Ala40, Thr45, Glu119, Phe184 and Ser201), and three others (Val115, Thr195 and Thr359) are located within a second residue shell not in direct contact with the substrate. The design of the library, described in the Supplementary Note 4, took account of natural sequence variation in the active-site region of Ec-LysC homologues and the results of computational re-design at nine residue positions in the vicinity of residue position 119. Restrictions placed on the number of permitted mutations at each of the eight residue positions in the library constrained its overall size to 2,160 possible theoretical combinations (Supplementary Table 5). A miniaturized screening protocol (see Methods) was devised to permit the direct measurement of ( L )-malate kinase activity by a single end-point measurement with an accuracy of ±8% in microtiter plates. To ensure adequate sequence space coverage 24 , 6,720 clones were tested by this method leading to the identification of nine positive variants which were confirmed by enzymatic assay of sequenced and individually purified clones. The best mutant Ec-LysC V115A:E119S:E434V exhibited a kcat/Km value of 0.82 s −1 mM −1 on ( L )-malate, approximately only twofold lower than that of the wild-type enzyme acting on the ( L )-aspartate natural substrate. In addition, the mutant retained very little activity towards ( L )-aspartate, resulting in a marked change in enzyme specificity (Fig. 2b). It is of note that the Glu434 position at the enzyme surface was not targeted in the construction of the library, but an E434V mutation was unexpectedly found in the best malate kinase mutant. A molecular model of ( L )-malate bound in the active-site of the ternary complex with the Ec-LysC V115A:E119S double mutant and Mg-ADP is shown in Fig. 2c.

To render the malate kinase enzyme more efficient for in vivo applications, we individually tested E250K, T344M, S345L and T352I mutations previously shown to alleviate feedback inhibition by lysine in the wild-type enzyme 25 . We found that all these mutations strongly decreased the inhibitory effect of lysine on malate kinase activity (Supplementary Fig. 4). The quadruple mutant, Ec-LysC V115A:E119S:E250K:E434V was therefore selected for implementation of the DHB pathway.

Engineering of malate semialdehyde dehydrogenase activity

The aspartate semialdehyde dehydrogenase from E. coli (Ec-Asd) enzyme was found to possess only trace activity on malyl-P in the reductive (biosynthetic) reaction direction, and on malate semialdehyde (MSA) in the reverse oxidative phosphorylation reaction (Supplementary Table 6). We first sought to engineer increased activity towards the malyl-P/MSA substrate/product couple through site-directed mutagenesis of the Ec-Asd Gram-negative bacterial enzyme. Active site residues involved in the binding of aspartate semialdehyde (ASA) to Ec-Asd have been previously identified in an X-ray crystal structure of a covalent complex formed as the reaction product of Cys135 thiol group attack on the substrate analogue S-methylcysteine sulfoxide in the presence of NADP + (ref. 26). The binding of the ASA α-amino and α-carboxylate groups in the Ec-Asd active-site occurs via salt-bridge interactions with oppositely charged Glu241 and Arg267 residue side-chains. This salt bridging arrangement is similar to that of the ( L )-aspartate substrate α-amino and α-carboxylate groups in the complex with E. coli aspartate kinase III (ref. 22) that catalyses the preceding reaction step in the physiological pathway. The 2-OH group in a malyl-P/MSA substrate/product couple might be expected to hydrogen bond with Glu241 in Ec-Asd thereby providing for substrate binding similar to that of the natural substrate derivative in the experimental complex. However, the poor observed activity of wild-type Ec-Asd on MSA compared to ASA may be in part due to a lowering in the binding affinity for an alternative substrate carrying net negative charge. Replacement of the conserved Glu241 residue in the wild-type E. coli enzyme by residues with uncharged side-chains would then be expected to improve MSA binding affinity and reduce that of ASA.

To test this hypothesis, saturation mutagenesis of Ec-Asd was carried out at residue position 241. Since aspartyl-P and malyl-P are highly unstable molecules, initial kinetic characterization of the wild-type and mutant enzymes was carried out on the MSA substrate of the reverse (oxidative phosphorylation) reaction. The wild-type enzyme and the most promising mutants (E241Q and E241C, see Supplementary Fig. 5) were then kinetically characterized in the biosynthetic (physiological) reaction direction in coupled enzyme reaction assays, using MK or AK to generate an in situ supply of malyl-P or aspartyl-P as appropriate. We found that the introduction of mutations E241Q and E241C respectively improved enzyme specificity by 71- and 17-fold in favour of the malyl-P substrate (Fig. 3a). However, the change in specificity was brought about by a marked decrease in activity towards the natural substrate aspartyl-P, rather than an intrinsic increase in activity towards malyl-P (Fig. 3b Supplementary Table 6). This result showed on one hand that the electrostatic interaction between the negatively charged Glu241 and the positively charged amino group of the natural aspartyl-P substrate was a major requirement for the wild-type activity of this enzyme. On the other hand, it became clear that MSD activity in Ec-Asd could not be significantly increased by simply imposing more favourable polar interactions between the α-hydroxyl group of malyl-P and alternative amino-acid residues in position 241. The active-site region in a modelled structure of a putative hemithioacetal MSA substrate derivative covalently bound to Cys135 in the Ec-Asd E241Q mutant (see Methods) is shown in Fig. 3c. The figure highlights electrostatic interactions between residues in the mutant enzyme active site and the covalently bound MSA reaction intermediate.

(a) Specificity (expressed as the ratio of maximum activity on aspartyl-P and malyl-P) of wild-type and mutant ASD enzymes from E. coli (Ec), B. subtilis (Bs), and M. jannaschii (Mj). (b) Activity of these enzymes on aspartyl-P and malyl-P. The results are the mean of at least two biological replicate experiments. Error bars correspond to the standard deviation of the mean. (c) Putative hemithioacetal malate semialdehyde (MSA) tetrahedral covalent reaction intermediate attached to Cys135 in the active-site of a computer-built model of an E241Q mutant of E. coli aspartate semialdehyde dehydrogenase (Ec-Asd) quaternary complex with non-covalently bound NADP(H) co-enzyme and inorganic phosphate (Pi). Carbon atoms in stick representations of enzyme residues, MSA reaction intermediate and the co-enzyme are respectively coloured in grey, green and cyan. Other atoms are shown in blue (nitrogen), red (oxygen), yellow (sulfur) and orange (phosphorus). Hydrogen bond interactions are represented as dashed-line vectors.

In an attempt to further increase malyl-P reductive dephosphorylation activity, orthologues of the ASD family from the Gram negative, and archaeal and fungal phylogenetic branches were examined as alternative enzyme engineering platforms. ASDs from these phylogenetic branches are differentiated by the presence of characteristic structural insertions and deletions in the co-enzyme binding-site region and at the enzyme homodimer subunit interface. Although a high degree of conservation of amino-acid residue functional groups exists within the active-site core, shared sequence identities of ASD family enzymes fall to as low as 10% (ref. 27). This structural variation correlates closely with marked differences in ASA oxidative phosphorylation catalytic efficiency, which varies by two orders of magnitude 28 . The natural variation in structure and sequence afforded by Gram-positive bacterial and archaeal ASDs may thus provide opportunities to modulate kinetic reaction rates other than through the introduction of additional mutations in the enzyme active-site.

The activities of wild-type and mutant ASDs from the Gram-positive bacterium Bacillus subtilis (Bs), that shares 26% sequence identity with Ec-Asd, and the archaeon Methanocaldococcus jannaschii (Mj) which is 21% identical to Ec-Asd, were assayed on aspartyl-P and malyl-P. Enzymatic activities of wild-type Bs-Asd and Mj-Asd on malyl-P were approximately threefold lower than those observed for Ec-Asd (Fig. 3b Supplementary Table 6). However, by analogy to the effects observed in Ec-Asd the reaction specificity of these enzymes is significantly improved in favour of malyl-P when the conserved active-site glutamate residues (Glu218 in Bs-Asd, Glu210 in Mj-Asd) are mutated to glutamine or cysteine. The best result was obtained for the Bs-Asd E218Q mutant enzyme, which displayed an almost eightfold increased activity on malyl-P, and a kcat value of 0.31 s −1 . This is comparable with Mj-Asd wild-type activity towards aspartyl-P (kcat=0.38 s −1 ). It is, however, much lower than that of the Bs-Asd wild-type (kcat =14.5 s −1 ). The kinetically efficient alternating-sites mechanism operating in bacterial ASD forms is absent in Mj-ASD and other archaeal ASDs 28,29 . Comparison of the relative turnover numbers suggests that the conversion of malyl-P to MSA catalysed by the Bs-Asd E218Q mutant may also be rate-limited by decoupled inter-subunit communication. Taking into account the loss in activity on the natural substrate, the specificity of Bs-Asd E218Q was shifted 650-fold towards malyl-P (Fig. 3a,b Supplementary Table 6). This enzyme variant was therefore chosen for integration in the construction of the DHB pathway.

Engineering of malate semialdehyde reductase activity

Having found homoserine dehydrogenase from Saccharomyces cerevisiae (Sc-Hom6) to be inactive on MSA, we sought to identify an alternative enzyme template for the engineering of MSR activity. We experimentally screened for MSA reductase activity in oxidoreductases that act on substrates structurally similar to MSA (Supplementary Table 7). Significant MSA reductase activity was detected for the broad-range aldehyde reductase, Ec-YqhD 30 , from E. coli, methylbutyraldehyde reductase, Sc-Ypr1 (ref. 31), from S. cerevisiae, 4-hydroxybutyrate dehydrogenase, Pg-4hbd 32,33 , from Porphyromonas gingivalis, and the succinic semialdehyde dehydrogenase, Ms-Ssr 34 , from Metallosphaera sedula. Amongst the enzymes that were active on MSA, the NADP + -dependent succinic semialdehyde reductase from Metallosphaera sedula 34 (Ms-Ssr) displayed the highest specific activity (4.0 μmol min −1 mg −1 ) and the best affinity (1.1 mM). However, its kcat/Km value was 112-fold lower than that of the (Sc-Hom6) homoserine dehydrogenase benchmark enzyme in the natural pathway on aspartate semialdehyde (Supplementary Table 7 Supplementary Note 5). Therefore, a comparative molecular modelling approach based on the exploitation of conserved relational structural and functional features was used to improve the catalytic efficiency of Ms-Ssr towards MSA.

Ms-Ssr is a zinc-dependent alcohol dehydrogenase 34 belonging to the medium-chain dehydrogenase/reductase (MDR) alcohol dehydrogenase (ADH) superfamily 35 . A molecular model of the Ms-Ssr dimer in a ternary complex with NADP + and ( L )-2,4-dihydroxybutyrate (DHB) was constructed using the atomic co-ordinate data from experimentally determined alcohol dehydrogenase structures (see Methods). Binding interactions of the zinc-coordinated DHB alcoholate anion in the active-site region are shown in Fig. 4a. The main contacts of the DHB α-carboxyl and 2-hydroxyl groups in the model are with the Gln108 and Asn305 residues. The presence of bulky Phe85 and Leu281 side-chains lining the substrate binding pocket suggests that the Ms-Ssr enzyme has an intrinsic preference for (unbranched) primary alcohol substrates. Structural analysis furthermore revealed that the enzyme employs a proton relay pathway which is different from the archetypal pathway that is present in most of the ADH family enzymes, and which is considered to be less efficient 35,36,37 (Fig. 4b). The engineering of an ADH1E-like proton relay system into Ms-Ssr, comprising a histidine at position 43 and an arginine at position 39, was thus considered to be a promising means of directly improving Ms-Ssr catalytic activity.

(a) Active-site region in a modelled complex of zinc-bound alcoholate ion of 2,4-dihydroxybutyric acid (DHB) with M. sedula succinic semialdehyde reductase (Ms-Ssr) and NADP + co-enzyme. Carbon atoms in stick representations of enzyme residues, DHB and the co-enzyme are respectively coloured in grey, green and cyan. Other atoms are coloured according to element type: nitrogen, blue oxygen, red sulfur, yellow phosphorus orange. Dashed lines indicate hydrogen bond interactions. The Zn 2+ ion is shown as a space filling sphere in grey. (b) Alternative proton relay systems operating in horse liver alcohol dehydrogenase (ADH1) and Ms-Ssr enzyme homologues. The overlay shows topologically equivalent residue positions in the modelled complex of Ms-Ssr and the zinc-bound DHB alcoholate ion (grey coloured carbon atoms) and the X-ray crystal structure of the horse liver ADH1 F93W mutant ternary complex (PDB code 1axe) with NAD + and trifluoroethanol (ETF) inhibitor (carbon atoms in green). Proton relay shuttles in the two enzymes are depicted as correspondingly grey and green coloured inter-atomic dashed-line connecting vectors. Other atoms are coloured according to element type as in (a), with fluorine atoms in ETF additionally shown in purple. The Ms-Ssr wild-type proton relay can be interchanged with the archetypal ADH1 shuttle in the Ms-Ssr H39R:N43H double mutant. (c) Kinetic parameters for Ms-Ssr mutants on succinic (SSA) and malic (MSA) semialdehyde. The results are the mean of at least three replicate experiments. Error bars correspond to the s.d.

The Ms-Ssr H39R:N43H double mutant exhibited an approximately fourfold increased catalytic efficiency on MSA compared to the wild-type enzyme, but still retained a strong preference for SSA over MSA (Fig. 4c, Supplementary Table 8). However, SSA is produced in E. coli only under extreme acid stress conditions, and its supply can be turned off by deleting both glutamate decarboxylases GadA and GadB 38 . Potential competition between SSA and MSA was therefore not considered to be a significant impediment to the efficient biosynthesis of DHB, and the Ms-Ssr H39R:N43H enzyme was thus chosen for incorporation in the DHB pathway.

Minimal metabolic engineering enables biosynthesis of DHB

To assure functional expression of the DHB pathway, we assembled a synthetic operon in the medium copy number pACT3 plasmid 39 , which expressed the three genes encoding the best DHB pathway enzymes (MK: Ec-LysC V115A:E119S:E250K:E434V, MSD: Bs-Asd E218Q, MSR: Ms-Ssr H39R:N43H) under the control of the inducible tac promoter. The resulting plasmid pDHB was transformed into the wild-type E. coli MG1655 strain and produced 60 mg l −1 DHB after 24 h of shake-flask cultivation in glucose mineral medium. The wild-type control strain that expressed the corresponding homoserine pathway enzymes (lysine resistant AK: LysC E250K, ASD: Ec-Asd, HSD: Sc-Hom6) produced no detectable quantities of DHB (Fig. 5 Supplementary Table 9). These results indicated that cellular DHB production via the synthetic pathway can be achieved, and we set-out to increase DHB production by metabolic engineering of the host strain and by optimization of the expression system.

Cells were cultivated in shake flasks on mineral medium containing 20 g per l glucose. Values correspond to concentrations after 24 h of cultivation. Error bars represent STDV from at least two replicate experiments. All strains were derived from E. coli K-12 substr. MG1655. All plasmids were derived from the pACT3 medium-copy number plasmid. pHOM expresses the genes encoding the homoserine pathway enzymes AK: Ec-LysC E250K, ASD: Ec-Asd, HSD: Sc-Hom6. pDHB expresses the genes encoding the DHB pathway enzymes MK: Ec-LysC V115A:E119S:E250K:E434V, MSD: Bs-Asd E218Q, MSR: Ms-Ssr H39R:N43H. pDHB-ppc* additionally expresses the malate-insensitive PEP carboxylase mutant PpcK620S. pDHBopt-ppc* has optimized ribosome binding sites in front of each DHB pathway gene. pDHBopt-ppc*(Ec-asd*) and pDHBopt-ppc*(Mj-asd*) express, respectively, the Ec-AsdE241Q or Mj-AsdE210Q mutant enzymes instead of Bs-AsdE218Q.

The DHB-producing strain accumulated 50% more acetate than the control strain (Fig. 5). Therefore, we first tested whether DHB production could be increased by inactivating the pyruvate oxidase (PoxB) 40 , or the acetate kinase/phosphate acetyltransferase (AckA-Pta)-dependent 41 acetate pathways. The deletion of poxB and ackA-pta alone or in combination significantly reduced the production of acetate (Fig. 5). DHB production after 24 h increased to 0.3 g l −1 and 0.4 g l −1 in the ΔackA-pta and ΔpoxB mutants, respectively. The deletion of both acetate pathways did not further improve strain performance and yielded 0.27g l −1 DHB (Fig. 5, Supplementary Table 9). Furthermore, conversion of malate into DHB by the synthetic pathway reduces the regeneration of oxaloacetate via the Krebs cycle. The resulting imbalance between oxaloacetate and acetyl-CoA production therefore may have led to the conversion of excess acetyl-CoA into acetate. To increase the availability of oxaloacetate, we overexpressed the anaplerotic phosphoenolpyruvate carboxylase (Ppc) enzyme. To prevent inhibition of the Ppc activity by aspartate or malate 42 , we employed the Ppc K620S mutant which is insensitive to feedback inhibition by the two compounds 43 and cloned the corresponding gene into the DHB operon resulting in plasmid pDHB-ppc*. When this plasmid was expressed in a wild-type strain, 1l DHB was produced after 24 h of cultivation and only trace amounts of acetate were detectable (Fig. 5). This result lends weight to the idea that a stoichiometric imbalance between the production of oxaloacetate and acetyl-CoA was indeed the major cause of increased acetate production in cells expressing the pDHB plasmid.

To improve the expression of the DHB pathway enzymes, their individual ribosome binding sites (RBS) were optimized 44 . The operon with optimized RBS sequences was assembled by fusion PCR and cloned downstream of the ppcK620S gene whose RBS was not changed since moderate overexpression of Ppc has been previously reported to be more beneficial for the production of Krebs cycle-derived C4 acids than maximizing Ppc activity 45,46,47 . When the optimized pDHBopt-ppc* plasmid was expressed in a wild-type strain, we observed the accumulation of 1.8 g l −1 DHB after 24 h of cultivation, corresponding to a molar yield of 0.15 (Fig. 5 Supplementary Table 9). This nearly twofold improvement can be attributed to a significant increase of MK and MSD activities, whereas the Ppc and MSA reductase activities remained nearly unchanged upon expression of the new plasmid (Supplementary Table 10). Expression of the optimized plasmid in the acetate pathway mutants did not further increase DHB production (Supplementary Table 9), in accord with the absence of acetate accumulation in DHB-producing strains. Furthermore, DHB synthesis dropped to ∼ 0.7 g l −1 when Mj-Asd E210Q or Ec-Asd E241Q were applied as the MSD enzyme, indicating that the superior in vitro performance of Bs-Asd E218Q also translates into the best cellular DHB production (Fig. 5). Taken together our results show that overexpression of the anaplerotic Ppc activity is a key factor in enabling cellular DHB production, and that increasing the barely measurable MSD activity is a major requirement for the further improvment of DHB production levels.

Mitochondria oxidize substrates to generate the ATP that fuels muscle contraction and locomotion. This review focuses on three steps in oxidative phosphorylation that have independent roles in setting the overall mitochondrial ATP flux and thereby have direct impact on locomotion. The first is the electron transport chain, which sets the pace for oxidation. New studies indicate that the electron transport chain capacity per mitochondria declines with age and disease, but can be revived by both acute and chronic treatments. The resulting higher ATP production is reflected in improved muscle power output and locomotory performance. The second step is the coupling of ATP supply from O2 uptake (mitochondrial coupling efficiency). Treatments that elevate mitochondrial coupling raise both exercise efficiency and the capacity for sustained exercise in both young and old muscle. The final step is ATP synthesis itself, which is under dynamic control at multiple sites to provide the 50-fold range of ATP flux between resting muscle and exercise at the mitochondrial capacity. Thus, malleability at sites in these subsystems of oxidative phosphorylation has an impact on ATP flux, with direct effects on exercise performance. Interventions are emerging that target these three independent subsystems to provide many paths to improve ATP flux and elevate the muscle performance lost to inactivity, age or disease.

Mitochondria are the powerhouses of biological tissues. In muscles, they link oxidation of substrates to phosphorylation that generates ATP. The contractile fibers then use ATP to generate force and motion. The role of mitochondria as the terminal sink for O2 in the respiratory system that sets the limit to maximum O2 uptake at the muscle level is well established (Weibel et al., 1991). A causal pathway linking this oxidation to the phosphorylation that generates ATP to fuel muscle force production and exercise performance is also clear. However, less clear is the direct role that mitochondria play in setting the limits to exercise performance. A few studies have made this connection in human subjects using exercise-training experiments, which are well known for raising the capacity for ATP supply (Jubrias et al., 2001) and mitochondrial volume density (Hoppeler et al., 1985). These studies show that a direct increase in muscle performance results from the greater energy supply capacity after endurance training. Thus mitochondria provide the bridge between the pathways that delivery oxygen and the synthesis of ATP that fuels muscle contraction and exercise performance.

One path to elevate ATP supply to raise exercise performance is to increase the mitochondrial content of muscle, as is typically found in endurance training of young subjects (Hoppeler et al., 1985). A second path is to improve the capacity for ATP generation per mitochondrion by targeting the processes underlying ATP supply. One such mechanism is the coupling of oxidation to phosphorylation (mitochondrial coupling efficiency), which can be improved to elevate ATP generation per O2 uptake. An example of this improvement is the acute effect of dietary nitrate on mitochondrial coupling efficiency, with direct effects on exercise efficiency in humans (Jones, 2014 Larsen et al., 2007). Free nitrate is released in the muscle cell by drinking beetroot juice, and acts via the nitrous oxide pathway to elevate mitochondrial ATP synthesis per O2 uptake (often expressed divided by 2 to yield the biochemical term of P/O). After drinking beetroot juice, the human subjects showing the largest increase in P/O had a correspondingly elevated exercise efficiency (leg power output per O2 uptake) on a cycle ergometer (Larsen et al., 2007). This link between mitochondrial and exercise efficiencies is the predicted response based on the thermodynamic connection between these processes (Whipp and Wasserman, 1969). Thus it is possible to adjust the underlying processes in oxidative phosphorylation to improve exercise performance.

A second example of the malleability of oxidative phosphorylation comes from the rapid effect of an antioxidant targeted to the mitochondrion, SS-31, on the capacity of mitochondria to generate ATP. A 1 h infusion of SS-31 in old mice raised P/O by 50% but doubled the phosphorylation capacity (ATPmax) in vivo in hindlimb muscles (Siegel et al., 2013). This greater rise in ATPmax than in P/O implies not only improvement in mitochondrial coupling efficiency but also a rapid rise in the capacity for electron transport chain (ETC) flux (O2 uptake capacity). The mechanism for this increase in the ETC flux capacity is thought to be stabilization of cardiolipin on the inner mitochondrial membrane, thereby restoring a key bridge in electron flow through the ETC (Birk et al., 2014 Szeto, 2014). These treatments demonstrate that there are many sites in oxidative phosphorylation that are potential targets for treatment to improve mitochondrial function. What is remarkable about beetroot juice and SS-31 is their speed of action in raising ATP supply capacity and exercise performance (1 h!). In contrast, many months of exercise training are needed to achieve the same goal (Jubrias et al., 2001). Thus multiple sites in oxidative phosphorylation have the potential to elevate energy supply and exercise performance – very rapidly with some treatments – without increasing the mitochondrial pool.

In this review I evaluate the three major steps in oxidative phosphorylation for malleability (Fig. 1) (Nicholls and Ferguson, 2002) that can improve exercise performance. The first step is the ETC, which oxidizes NADH to pump H + to generate a proton motive force across the inner mitochondrial membrane. The second step uses the proton motive force to drive phosphorylation via the F1F0-ATP synthase. The third step involves short-circuiting the H + gradient via a number of processes that leak H + through the inner mitochondrial membrane, thereby circumventing phosphorylation. My goal is to evaluate how each step contributes to ATP production and thereby has impact on exercise performance. The exciting implication of these insights is that mitochondria have multiple sites of malleability that provide targets for optimizing function to improve ATP flux and elevate muscle exercise performance in both healthy and diseased states.

Materials and methods

Cell lines and culture conditions

Immortalized baby mouse kidney (iBMK) epithelial cells were generated as described previously ( Degenhardt et al, 2002 ). Briefly, primary kidney epithelial cells from mice double deficient for Bax and Bak (Bax −/− /Bak −/− ) were immortalized by E1A and dominant-negative p53 expression ( Degenhardt et al, 2002 Mathew et al, 2008 ). iBMK cells expressing human oncogenic H-Ras V12G or myr-Akt were derived by electroporation with pcDNA1.H-Ras V12G ( Lin et al, 1995 ) or pcDNA3.myr-Akt ( Plas et al, 2001 ), respectively, followed by zeocin selection. The resulting cell lines were grown in Dulbecco's modified eagle media (DMEM) without pyruvate (Cellgro), supplemented with 10% dialyzed fetal bovine serum (HyClone) in all metabolomics experiments. For normoxia experiments, cells are grown in an incubator containing 5% CO2 and ambient oxygen at 37°C. For hypoxia experiments, cells are grown and all experiments are completed inside a hypoxia glove box (Coy Lab) containing 1% oxygen and 5% CO2 at 37°C. For labeling experiments, medium was prepared from DMEM without glucose or glutamine (Cellgro), with the desired isotopic form of glucose and/or glutamine added to a final concentration of 4.5 g/l glucose and 0.584 g/l glutamine. Short-term experiments (e.g., nutrient uptake and kinetic flux profiling) were conducted at 70–80% confluency for longer-term labeling experiments, confluency varied as the cells multiplied.

Exchange rate measurements

Media samples were collected at various time points. Glucose, glutamine, and lactate were measured by enzymatic assay with electrochemical detection on a YSI7200 instrument (YSI, Yellow Springs, OH). Alanine and pyruvate were measured by LC-MS. Oxygen consumption was measured using a Seahorse XF24 flux analyzer (Seahorse Bioscience, North Billerica, MA). To measure oxygen uptake in hypoxia, the Seahorse instrument was placed in the hypoxia chamber with 1% oxygen. To probe the fraction of oxygen consumption, which is effectively coupled with the electron transport chain, we measured the oxygen consumption rate when cells were treated with an electron transport chain inhibitor, antimycin A. We observed that ∼80% of oxygen consumption across the studied cell lines and growth conditions was used for oxidative phosphorylation.

Metabolomic experiments and LC-MS analysis

For all metabolomic and isotope-tracer experiments, metabolism was quenched, and metabolites were extracted by quickly aspirating the media and immediately adding a 80:20 methanol:water extraction solution at −80°C.

Samples were analyzed using multiple LC-MS systems (each from Thermo Scientific and fed by electrospray ionization), as described previously ( Munger et al, 2008 Lemons et al, 2010 Lu et al, 2010 ). In brief, a stand-alone orbitrap mass spectrometer (Exactive) operating in negative-ion mode was coupled to reversed-phase ion-pairing chromatography and used to scan from m/z 85–1000 at 1 Hz and 100 000 resolution a TSQ Quantum Ultra triple-quadrupole mass spectrometer operating in positive-ion mode was coupled to hydrophilic interaction chromatography on an aminopropyl column and used to analyze selected compounds by multiple reaction monitoring and a TSQ Quantum Discovery triple-quadrupole mass spectrometer operating in negative-ion mode was coupled to reverse-phase ion-pairing chromatography and used to analyze selected compounds by multiple reaction monitoring. Data were analyzed using the MAVEN software suite ( Melamud et al, 2010 ). The results are adjusted for natural 13 C abundance and enrichment impurity of labeled substrate supplied to cells.

Absolute metabolite levels were quantified as previously described ( Bennett et al, 2008 ) and normalized by packed cell volume.

De novo serine synthesis rate

To quantify the rate of serine synthesis, cells were cultured in DMEM media containing U- 13 C-serine. The steady-state labeling pattern of intracellular 3-phosphoglycerate, serine, and glycine was measured by extracting metabolites after washing three times with ice-cold PBS. Labeled 3-phosphoglycerate was never observed, confirming that reverse serine synthesis pathway flux is negligible. Glycine was observed in two forms: unlabeled and M+2 (whose relative fractions are referred to as G0 and G2 below). Serine was observed in four forms: unlabeled, M+1, M+2, and M+3, with fractions S0, S1, S2, S3, respectively. These forms arise via de novo synthesis from 3-phosphoglycerate (making unlabeled via F1, see Supplementary Figure 9), uptake from media (making M+3 via F2), and reverse SHMT flux from glycine (making all possible forms, depending on the labeling of glycine and the methylene-THF methyl group, via F3). The fraction of methylene-THF with the reactive one-carbon unit unlabeled is denoted T0 and that labeled on the one-carbon unit is denoted T1. We also measured net serine uptake flux F2 by monitoring the change of serine concentration in the media.

Under isotopic steady state, the balance equations for the four labeling forms of serine can be formulated as following:


Discovering motifADE. Systematic identification of transcription factors involved in biological processes in mammals remains a largely unsolved problem (17). A promising approach relates genome-wide expression profiles to promoter sequences to discover influential cis motifs (18�). Such methods have yielded impressive results in simple organisms such as yeast, but it has been challenging to extend these algorithms to mammalian genomes, where intergenic regions are large, annotation of gene structure is imperfect, and DNA sequence can be highly repetitive. Most of these methods seek motifs by comparison with a fixed background model of nucleotide composition (which fails to represent the fluctuations seen in large genomes) or by comparison between two sets of genes (which is likely to capture only very sharp differences). Furthermore, many of these methods assume that the expression data are normally distributed, which may not always be true.

To overcome some of these obstacles, we devised a simple, nonparametric strategy for identifying motifADE ( Fig. 1A ). The algorithm involves three steps: (i) ranking genes based on differential expression between two conditions (ii) given a candidate motif, identifying the subset of genes whose promoter regions contains the motif and (iii) testing by means of a nonparametric, rank sum statistic whether these genes tend to appear toward the top or bottom of the ranked list (indicating association) or are randomly distributed on the list. motifADE may be applied to a specific candidate motif of interest or to the list of all possible motifs of a given size (in which case the significance level should be adjusted to reflect multiple hypothesis testing). By using a nonparametric scoring procedure, we do not make assumptions about the distribution of the expression data. Furthermore, by considering the entire rank-ordered list, the promoters without the motif implicitly provide a background of DNA composition for comparison, and there is no need to demarcate or cluster a group of genes as being “responsive.” The method can operate on a traditional promoter database or even a database of promoters that have been masked based on evolutionary conservation.

Schematic overview of motifADE and application to the PGC-1α time course. (A) The motifADE strategy. It begins with a list of genes ordered on the basis of differential expression across two conditions. Each gene is then annotated for the presence of a given motif in the promoter region. A nonparametric statistic is used to assess whether genes with the motif tend to rank high on this list. In this example, genes with Motif 1 are randomly distributed on the list, whereas genes with Motif 2 tend to rank high, suggesting an association between Motif 2 and the differential expression. (B) C2C12 cells were infected with an adenovirus expressing either GFP (control) or with PGC-1α and were profiled over a 3-day period. Experiments were performed in duplicate and relative gene expression measures are shown. Genes are ranked according to the difference in expression between PGC-1α and GFP on day 3. Mouse genes having a perfect Errα motif (5′-TGACCTTG-3′), a perfect Gabpa/b motif (5′-CTTCCG-3′), or both motifs are labeled with a black bar on the right side of the correlogram.

Binding Sites for Errα and Gabpa Are the Top-Scoring Motifs Associated with the PGC-1α Transcriptional Program. To identify motifs related to PGC-1α action, we infected mouse C2C12 muscle cells with an adenovirus expressing PGC-1α and obtained gene expression profiles for 12,488 genes at 0, 1, 2, and 3 days after infection. We found 649 genes that were induced at least 1.5-fold (nominal P < 0.05) at day 3. As expected, these genes were enriched for genes involved in carbohydrate metabolism and the mitochondrion (1). Interestingly, many genes involved with protein synthesis (gene ontology terms: protein biosynthesis, mitochondrial ribosome, and ribosome) are also induced.

We then applied motifADE to study the 5,034 mouse genes for which we have measures of gene expression, as well as reliable annotations of the TSS. For each gene, the target region was defined to be a 2-kb region centered on the TSS. We then tested all possible k-mers ranging in size from k = 6 to k = 9 nucleotides for association with differential expression on each of the 3 days of the time course. A total of 20 motifs achieved high statistical significance (P < 0.001, after Bonferroni correction for multiple hypothesis testing) and these were almost exclusively related to two distinct motifs ( Table 1 , and Table 2, which is published as supporting information on the PNAS web site). The first motif, 5′-TGACCTTG-3′ was significant on days 1, 2, and 3 (adjusted P = 2.1 × 10 -6 ,2.9 × 10 -9 , and 7.7 × 10 -7 , respectively). It corresponds to the published binding site for the orphan nuclear receptor Errα (22), which is known to be capable of being coactivated by PGC-1α and -1β (23�). The Errα gene is known to be involved in metabolic processes, based on studies showing that knockout mice have reduced body weight and peripheral fat tissue, as well as altered expression of genes involved in metabolic pathways (26). The second motif is 5′-CTTCCG-3′ (adjusted P = 8.9 × 10 -9 ), which is the top-scoring motif on day 3. It corresponds to the published binding site for Gabpa (27), which complexes with Gabpb (15) to form the heterodimer, nuclear respiratory factor-2 (NRF-2), a factor known to regulate the expression of some OXPHOS genes (28). Interestingly, the reverse complements of these motifs did not score as well, suggesting a preference for the orientation of these motifs, and some occurrences of the motifs were found downstream of the TSS. Whereas each of these motifs is individually associated with PGC-1α, our analyses suggest that a gene having both motifs typically ranks higher on the list of differentially expressed genes than genes with only one of the motifs (Fig. 6, which is published as supporting information on the PNAS web site), suggesting that the two motifs might have an additive or synergistic effect.

Table 1.

Day Motif Frequency Nominal P value Adjusted P value Annotation Ref.
1 TGACCTTG 0.07 3.15 × 10 -11 2.06 × 10 -6 Errα 22
TGACCTTGA 0.02 4.59 × 10 -10 1.20 × 10 -4 Errα
2 TGACCTTG 0.07 4.44 × 10 -14 2.91 × 10 -9 Errα 22
TGACCTT 0.16 3.62 × 10 -12 5.93 × 10 -8 Errα
TGACCT 0.45 1.46 × 10 -11 5.97 × 10 -8 NR half-site 34
GACCTTG 0.16 7.92 × 10 -11 1.30 × 10 -6 Errα
GACCTT 0.41 1.42 × 10 -9 5.81 × 10 -6 Errα
TTGACC 0.27 2.42 × 10 -7 9.92 × 10 -4 Errα
3 CTTCCG 0.33 2.19 × 10 -12 8.97 × 10 -9 Gabpa 35
TGACCTTG 0.07 1.17 × 10 -11 7.66 × 10 -7 Errα 22
TGACCTT 0.16 1.23 × 10 -10 2.02 × 10 -6 Errα
CCCGCC 0.54 2.04 × 10 -8 8.36 × 10 -5
GCGGCG 0.43 3.78 × 10 -8 1.55 × 10 -4
AGGTCA 0.42 3.90 × 10 -8 1.60 × 10 -4 NR half-site 34
CTTCCGG 0.16 1.95 × 10 -8 3.19 × 10 -4 Gabpa
TTCCGG 0.31 1.09 × 10 -7 4.46 × 10 -4 Gabpa
GGGGCG 0.54 1.24 × 10 -7 5.08 × 10 -4
TTCCGCT 0.07 3.30 × 10 -6 5.41 × 10 -4 Gabpa
GCCGGC 0.42 1.57 × 10 -7 6.44 × 10 -4
ACTTCCG 0.09 5.11 × 10 -8 8.38 × 10 -4 Gabpa

motiFADE was performed by using the mouse promoter database on each of days 1, 2, and 3. All motifs achieving a Bonferroni-corrected P value < 1 × 10 -3 are shown. Annotations of the motif and the literature references, when available, are indicated. NR, nuclear receptor.

Errα and Gabpa Motifs Are Evolutionarily Conserved and Enriched Upstream of OXPHOS Genes. We next repeated motifADE analysis by using a “masked” promoter database (Table 3, which is published as supporting information on the PNAS web site). We still considered the 2,000 base pairs centered on the TSS, but we only considered those nucleotides aligned and conserved between mouse and human. Still, the top ranking motifs on days 1 and 3 were related to Errα (adjusted P = 4.8 × 10 -6 , P = 1.2 × 10 -11 , respectively) and to Gabpa (day 3 P = 3.1 × 10 -11 ), providing additional support that these motifs are biologically relevant.

The Errα and Gabpa motifs are particularly enriched upstream of a coregulated subset of OXPHOS genes, which exhibit reduced expression in human diabetes (5, 6). Whereas the top-scoring Errα motif (5′-TGACCTTG-3′ or its reverse complement) only occurs in 12% of the promoters in the database, in 29% of the PGC-responsive genes (i.e., those genes induced at least 1.5-fold on day 3), and in 27% of the mitochondrial genes, they are found in 52% of the coregulated subset of OXPHOS genes (significance of enrichment, P = 1 × 10 -4 ). Approximately one-half of these sites are perfectly conserved in the syntenic region in human (data not shown). The top-scoring Gabpa-binding sites (5′-CTTCCG-3′ or its reverse complement) are much more common (62% of all promoters of the database and in 79% of the PGC-responsive genes), however, they too show significant enrichment in the coregulated subset of OXPHOS genes (89%, P = 0.02).

Errα and Gabpa Are Themselves Induced by PGC-1α. The above results suggest that Errα and Gabpa may be the key transcriptional factors mediating PGC-1α action in muscle. In this connection, it is notable that based on the microarray data, both Errα and Gabpa are themselves induced 𢒂-fold (P < 0.01) on day 1 after expression of PGC-1α, which is consistent with previous studies (2, 23). Moreover, careful analysis of the Errα and Gabpa genes suggest that each contain potential binding sites for both transcription factors within the vicinity of their promoters. The Errα gene has the Errα motif, as well as a conserved variant of the Gabpa-binding site (27) upstream of the TSS, whereas the Gabpa gene has an Errα site upstream of the TSS and a conserved variant of the Gabpa-binding site in its first intron. These results raise the possibility that Errα and Gabpa may regulate their own expression, and that of each other.

A Transcriptional Switch Driving Mitochondrial Biogenesis. Taken together, the systematic analysis of the transcriptional program driven by PGC-1α in skeletal muscle suggests a model ( Fig. 2 ) in which increases in PGC-1α protein levels results in increased transcriptional activity of Gabpa and Errα on their own promoters, leading to a stable increase in the expression of these two factors by means of a double-positive-feedback loop. These two factors, perhaps in combination with PGC-1α, are then crucial in the induction of downstream target genes, many of which have binding sites for these motifs ( Fig. 2 ). Such a circuit may serve as a regulatory switch, analogous to a feed-forward loop that plays a key role in the early stages of endomesodermal development in sea urchin (29).

Proposed model of mechanism of action of PGC-1α. PGC-1α is a highly regulated gene that responds to external stimuli, e.g., reduced in diabetes and increased after exercise. When PGC-1α levels rise, the expression of Errα and Gabpa are immediately induced by means of a double-positive-feedback loop. These levels rise, and over the course of 2𠄳 days, these factors couple with PGC-1α to induce the expression of NRF-1 and hundreds of downstream targets, such as OXPHOS and other mitochondrial genes that are enriched for these transcription factor-binding sites.

Experimental Validation of the Model. We experimentally tested this model of gene regulation. To determine whether Errα and Gabpa are indeed key transcriptional partners of PGC-1α, we tested whether they are coactivated by PGC-1α on the Errα and Gabpa promoters ( Fig. 3 ). We cloned appropriate portions of these promoters into expression vectors and tested these factors' ability to drive expression of a reporter gene, in the presence and absence of PGC-1α. The results confirm the predicted coactivation between PGC-1α and the two transcription factors in muscle cells. Moreover, the results for the Errα promoter fragment containing both sites show that the transcription factors act additively or even synergistically ( Fig. 3 ).

Errα and Gabpa cooperate with PGC-1α to induce their own expression. (A) Putative Errα and Gabpa motifs 1 kb upstream and downstream of the Errα and the Gabpa TSSs. (BD) C2C12 cells were transfected with a reporter gene plasmid containing 2 kb of the Errα promoter (B), Gabpa promoter (C), or Gabpa intron 1 (D), together with expression plasmids for Errα, Gabpa, Gabpb1, and PGC-1α. Forty-eight hours after transfection, reporter gene levels were determined and were normalized to β-galactosidase levels.

We next sought to explore the functional role of Errα in executing the PGC-1α-mediated response, by using a recently developed, selective Errα inverse agonist, called XCT790 (referred to below as the inhibitor Fig. 7, which is published as supporting information on the PNAS web site). This compound does not significantly inhibit or activate other nuclear receptors at doses near its IC50 (583 nM) for Errα, although higher concentrations (ϣ.3 μM) have been found to weakly activate PPARγ (R.A.H., personal communication). In an affinity chromatography assay, XCT790 specifically reduced the interaction between PGC-1α and Errα, whereas PGC-1α binding to another nuclear receptor, the hepatic nuclear factor 4α was not affected (Fig. 8, which is published as supporting information on the PNAS web site).

The inhibitor was first used to probe the role of Errα in activation of Errα and Gabpa, by repeating the above described experiments in the presence or absence of the inhibitor at a dose slightly above its IC50. The inhibitor markedly reduced activation of the Errα promoter by Errα and PGC-1α, but had no effect on Gabpa/b-driven reporter gene expression ( Fig. 4A ). The effect of the Errα inhibitor resembles the effect of site-directed mutagenesis of the Errα-binding site on the Errα promoter ( Fig. 4B ).

A small-molecule inhibitor of Errα inhibits the response to PGC-1α. (A) C2C12 myoblasts transfected with wild-type Errα promoter and the respective expression plasmids were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 48 h before reporter gene levels were measured. (B) C2C12 cells were transfected with wild-type or Errα-promoter harboring a mutated Errα motif (mutant), together with expression plasmids for Errα, Gabpa, Gabpb1, and PGC-1α. After 48 h, reporter gene levels were determined and normalized to β-galactosidase levels. (C) C2C12 myotubes were infected with GFP- or PGC-1α adenovirus and were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 1 day. Relative expression levels of several genes were then determined by semiquantitative real-time PCR and were normalized against 18S rRNA levels. (D) Fao rat hepatoma cells were infected with adenoviral GFP or PGC-1α and were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 1 day before relative gene expression levels were measured by real-time PCR, and were normalized against 18S rRNA levels. * , P < 0.05. NS, not significant.

The role of Errα was next investigated in the regulation of endogenous PGC-1α target genes. PGC-1α was expressed in C2C12 myotubes, treated with inhibitor or vehicle, and we measured the expression level of specific genes on day 1 by using RT-PCR. We studied five genes that contain variants of the Errα motif, including two of the coregulated subset of OXPHOS genes, Gabpa, and Errα, as well as medium-chain acyl-CoA dehydrogenase (MCAD), a published target of Errα (30). The expression of all of these genes is induced by PGC-1α, and this induction is diminished by the inhibitor ( Fig. 4C ). The specificity of the response was confirmed by using two genes that are induced by PGC-1α that lack obvious Errα-binding sites ( Fig. 4C ). Together, these results suggest that a subset of PGC-1α genes are regulated by Errα, primarily the target genes containing an Errα motif. The fact that the inhibition of these genes by the ERRα inverse agonist was not complete suggests that other factors might also be involved in this regulation. To ensure that the effects of the Errα inhibitor were not due to activation of PPARγ, control experiments were performed by using the synthetic PPARγ agonist rosiglitazone, and confirmed that it had no effect on the expression of these genes (Fig. 9, which is published as supporting information on the PNAS web site).

Errα Inhibition Reduces the PGC-1α Program in Cellular Assays. The role of Errα in the PGC-1α-mediated physiologic response was characterized by using functional assays of mitochondrial respiration. The Errα inhibitor potently diminishes PGC-1α induction of total cellular respiration and also significantly reduces PGC-1α-triggered increases in uncoupled respiration ( Fig. 5 ). The data suggest that inhibition of Errα elicits certain aspects of a diabetic phenotype in cultured muscle cells.

Total and uncoupled mitochondrial respiration are inhibited by the synthetic Errα-inverse agonist XCT790. (A and B) C2C12 myotubes were infected with GFP or PGC-1α adenovirus and were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 2 days before total mitochondrial respiration (A) and uncoupled respiration (B) was determined. * , P < 0.05 in paired t test.

Inhibition of Errα Does Not Affect Gluconeogenesis in Liver. We extended the analysis to cells derived from liver, where PGC-1α has been shown to induce genes of the fasting response, including those involved in gluconeogenesis and β-oxidation of fatty acids (1). These responses are generally elevated in poorly controlled diabetics. As expected, MCAD and two key genes related to gluconeogenesis (glucose-6-phosphatase and phosphoenol-pyruvate carboxykinase) were all induced by PGC-1α. However, the Errα antagonist repressed PGC-1α induction of MCAD but not the gluconeogenic genes ( Fig. 4D ).

NRF-1 Is Downstream of Errα and Gabpa. Finally, we investigated the relationship of NRF-1 to Errα function in muscle cells. NRF-1 has been suggested to be a key transcription factor involved in mito-chondrial biogenesis (31), and is induced and coactivated by PGC-1α in this process (2). However, what has been puzzling is that transgenic overexpression of NRF-1 in muscle is not sufficient to induce mitochondrial biogenesis (32). By using the synthetic Errα inverse agonist, we found that induction of NRF-1 by PGC-1α is actually downstream of the initial Errα-mediated events (Fig. 10, which is published as supporting information on the PNAS web site). Interestingly, motifADE analysis of the NRF-1 consensus motif (5′-GCGCAYGCGC-3′ or reverse complement) yields a score that is significant if considered as a test of single hypothesis (nominal P = 0.001 on day 3). Overall, these results suggest that NRF-1 is an important but relatively late mediator of the mito-chondrial transcriptional program induced by PGC-1α.

  • Dedication
  • About the authors
  • Preface
  • Acknowledgments
  • Part I. The Molecular Design of Life
    • Chapter 1. Prelude: Biochemistry and the Genomic Revolution
      • 1.1. DNA Illustrates the Relation between Form and Function
        • 1.1.1. DNA Is Constructed from Four Building Blocks
        • 1.1.2. Two Single Strands of DNA Combine to Form a Double Helix
        • 1.1.3. RNA Is an Intermediate in the Flow of Genetic Information
        • 1.1.4. Proteins, Encoded by Nucleic Acids, Perform Most Cell Functions
        • 1.3.1. Reversible Interactions of Biomolecules Are Mediated by Three Kinds of Noncovalent Bonds
        • 1.3.2. The Properties of Water Affect the Bonding Abilities of Biomolecules
        • 1.3.3. Entropy and the Laws of Thermodynamics
        • 1.3.4. Protein Folding Can Be Understood in Terms of Free-Energy Changes
        • Stereochemical Renderings
        • Fischer Projections
        • Key Terms
        • 2.1. Key Organic Molecules Are Used by Living Systems
          • 2.1.1. Many Components of Biochemical Macromolecules Can Be Produced in Simple, Prebiotic Reactions
          • 2.1.2. Uncertainties Obscure the Origins of Some Key Biomolecules
          • 2.2.1. The Principles of Evolution Can Be Demonstrated in Vitro
          • 2.2.2. RNA Molecules Can Act As Catalysts
          • 2.2.3. Amino Acids and Their Polymers Can Play Biosynthetic and Catalytic Roles
          • 2.2.4. RNA Template-Directed Polypeptide Synthesis Links the RNA and Protein Worlds
          • 2.2.5. The Genetic Code Elucidates the Mechanisms of Evolution
          • 2.2.6. Transfer RNAs Illustrate Evolution by Gene Duplication
          • 2.2.7. DNA Is a Stable Storage Form for Genetic Information
          • 2.3.1. ATP, a Common Currency for Biochemical Energy, Can Be Generated Through the Breakdown of Organic Molecules
          • 2.3.2. Cells Were Formed by the Inclusion of Nucleic Acids Within Membranes
          • 2.3.3. Compartmentalization Required the Development of Ion Pumps
          • 2.3.4. Proton Gradients Can Be Used to Drive the Synthesis of ATP
          • 2.3.5. Molecular Oxygen, a Toxic By-Product of Some Photosynthetic Processes, Can Be Utilized for Metabolic Purposes
          • 2.4.1. Filamentous Structures and Molecular Motors Enable Intracellular and Cellular Movement
          • 2.4.2. Some Cells Can Interact to Form Colonies with Specialized Functions
          • 2.4.3. The Development of Multicellular Organisms Requires the Orchestrated Differentiation of Cells
          • 2.4.4. The Unity of Biochemistry Allows Human Biology to Be Effectively Probed Through Studies of Other Organisms
          • Key Organic Molecules Are Used by Living Systems
          • Evolution Requires Reproduction, Variation, and Selective Pressure
          • Energy Transformations Are Necessary to Sustain Living Systems
          • Cells Can Respond to Changes in Their Environments
          • Key Terms
          • Where to start
          • Books
          • Prebiotic chemistry
          • In vitro evolution
          • Replication and catalytic RNA
          • Transition from RNA to DNA
          • Membranes
          • Multicellular organisms and development
          • 3.1. Proteins Are Built from a Repertoire of 20 Amino Acids
          • 3.2. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains
            • 3.2.1. Proteins Have Unique Amino Acid Sequences That Are Specified by Genes
            • 3.2.2. Polypeptide Chains Are Flexible Yet Conformationally Restricted
            • 3.3.1. The Alpha Helix Is a Coiled Structure Stabilized by Intrachain Hydrogen Bonds
            • 3.3.2. Beta Sheets Are Stabilized by Hydrogen Bonding Between Polypeptide Strands
            • 3.3.3. Polypeptide Chains Can Change Direction by Making Reverse Turns and Loops
            • 3.6.1. Amino Acids Have Different Propensities for Forming Alpha Helices, Beta Sheets, and Beta Turns
            • 3.6.2. Protein Folding Is a Highly Cooperative Process
            • 3.6.3. Proteins Fold by Progressive Stabilization of Intermediates Rather Than by Random Search
            • 3.6.4. Prediction of Three-Dimensional Structure from Sequence Remains a Great Challenge
            • 3.6.5. Protein Modification and Cleavage Confer New Capabilities
            • Proteins Are Built from a Repertoire of 20 Amino Acids
            • Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains
            • Secondary Structure: Polypeptide Chains Can Fold into Regular Structures Such as the Alpha Helix, the Beta Sheet, and Turns and Loops
            • Tertiary Structure: Water-Soluble Proteins Fold into Compact Structures with Nonpolar Cores
            • Quaternary Structure: Polypeptide Chains Can Assemble into Multisubunit Structures
            • The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure
            • Key Terms
            • Ionization of Water
            • Definition of Acid and Base
            • Definition of pH and pK
            • Henderson-Hasselbalch Equation
            • Buffers
            • pKa Values of Amino Acids
            • Media Problem
            • Where to start
            • Books
            • Conformation of proteins
            • Alpha helices, beta sheets, and loops
            • Domains
            • Protein folding
            • Covalent modification of proteins
            • Molecular graphics
            • 4.1. The Purification of Proteins Is an Essential First Step in Understanding Their Function
              • 4.1.1. The Assay: How Do We Recognize the Protein That We Are Looking For?
              • 4.1.2. Proteins Must Be Released from the Cell to Be Purified
              • 4.1.3. Proteins Can Be Purified According to Solubility, Size, Charge, and Binding Affinity
              • 4.1.4. Proteins Can Be Separated by Gel Electrophoresis and Displayed
              • 4.1.5. A Protein Purification Scheme Can Be Quantitatively Evaluated
              • 4.1.6. Ultracentrifugation Is Valuable for Separating Biomolecules and Determining Their Masses
              • 4.1.7. The Mass of a Protein Can Be Precisely Determined by Mass Spectrometry
              • 4.2.1. Proteins Can Be Specifically Cleaved into Small Peptides to Facilitate Analysis
              • 4.2.2. Amino Acid Sequences Are Sources of Many Kinds of Insight
              • 4.2.3. Recombinant DNA Technology Has Revolutionized Protein Sequencing
              • 4.3.1. Antibodies to Specific Proteins Can Be Generated
              • 4.3.2. Monoclonal Antibodies with Virtually Any Desired Specificity Can Be Readily Prepared
              • 4.3.3. Proteins Can Be Detected and Quantitated by Using an Enzyme-Linked Immunosorbent Assay
              • 4.3.4. Western Blotting Permits the Detection of Proteins Separated by Gel Electrophoresis
              • 4.3.5. Fluorescent Markers Make Possible the Visualization of Proteins in the Cell
              • 4.5.1. Nuclear Magnetic Resonance Spectroscopy Can Reveal the Structures of Proteins in Solution
              • 4.5.2. X-Ray Crystallography Reveals Three-Dimensional Structure in Atomic Detail
              • The Purification of Proteins Is an Essential Step in Understanding Their Function
              • Amino Acid Sequences Can Be Determined by Automated Edman Degradation
              • Immunology Provides Important Techniques with Which to Investigate Proteins
              • Peptides Can Be Synthesized by Automated Solid-Phase Methods
              • Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography
              • Key Terms
              • Chapter Integration Problems
              • Data Interpretation Problems
              • Where to start
              • Books
              • Protein purification and analysis
              • Ultracentrifugation and mass spectrometry
              • X-ray crystallography and spectroscopy
              • Monoclonal antibodies and fluorescent molecules
              • Chemical synthesis of proteins
              • 5.1. A Nucleic Acid Consists of Four Kinds of Bases Linked to a Sugar-Phosphate Backbone
                • 5.1.1. RNA and DNA Differ in the Sugar Component and One of the Bases
                • 5.1.2. Nucleotides Are the Monomeric Units of Nucleic Acids
                • 5.2.1. The Double Helix Is Stabilized by Hydrogen Bonds and Hydrophobic Interactions
                • 5.2.2. The Double Helix Facilitates the Accurate Transmission of Hereditary Information
                • 5.2.3. The Double Helix Can Be Reversibly Melted
                • 5.2.4. Some DNA Molecules Are Circular and Supercoiled
                • 5.2.5. Single-Stranded Nucleic Acids Can Adopt Elaborate Structures
                • 5.3.1. DNA Polymerase Catalyzes Phosphodiester-Bond Formation
                • 5.3.2. The Genes of Some Viruses Are Made of RNA
                • 5.4.1. Several Kinds of RNA Play Key Roles in Gene Expression
                • 5.4.2. All Cellular RNA Is Synthesized by RNA Polymerases
                • 5.4.3. RNA Polymerases Take Instructions from DNA Templates
                • 5.4.4. Transcription Begins near Promoter Sites and Ends at Terminator Sites
                • 5.4.5. Transfer RNA Is the Adaptor Molecule in Protein Synthesis
                • 5.5.1. Major Features of the Genetic Code
                • 5.5.2. Messenger RNA Contains Start and Stop Signals for Protein Synthesis
                • 5.5.3. The Genetic Code Is Nearly Universal
                • 5.6.1. RNA Processing Generates Mature RNA
                • 5.6.2. Many Exons Encode Protein Domains
                • A Nucleic Acid Consists of Four Kinds of Bases Linked to a Sugar-Phosphate Backbone
                • A Pair of Nucleic Acid Chains with Complementary Sequences Can Form a Double-Helical Structure
                • DNA Is Replicated by Polymerases That Take Instructions from Templates
                • Gene Expression Is the Transformation of DNA Information into Functional Molecules
                • Amino Acids Are Encoded by Groups of Three Bases Starting from a Fixed Point
                • Most Eukaryotic Genes Are Mosaics of Introns and Exons
                • Key Terms
                • Chapter Integration Problems
                • Media Problem
                • Where to start
                • Books
                • DNA structure
                • DNA replication
                • Discovery of messenger RNA
                • Genetic code
                • Introns, exons, and split genes
                • Reminiscences and historical accounts
                • 6.1. The Basic Tools of Gene Exploration
                  • 6.1.1. Restriction Enzymes Split DNA into Specific Fragments
                  • 6.1.2. Restriction Fragments Can Be Separated by Gel Electrophoresis and Visualized
                  • 6.1.3. DNA Is Usually Sequenced by Controlled Termination of Replication (Sanger Dideoxy Method)
                  • 6.1.4. DNA Probes and Genes Can Be Synthesized by Automated Solid-Phase Methods
                  • 6.1.5. Selected DNA Sequences Can Be Greatly Amplified by the Polymerase Chain Reaction
                  • 6.1.6. PCR Is a Powerful Technique in Medical Diagnostics, Forensics, and Molecular Evolution
                  • 6.2.1. Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant DNA Molecules
                  • 6.2.2. Plasmids and Lambda Phage Are Choice Vectors for DNA Cloning in Bacteria
                  • 6.2.3. Specific Genes Can Be Cloned from Digests of Genomic DNA
                  • 6.2.4. Long Stretches of DNA Can Be Efficiently Analyzed by Chromosome Walking
                  • 6.3.1. Complementary DNA Prepared from mRNA Can Be Expressed in Host Cells
                  • 6.3.2. Gene-Expression Levels Can Be Comprehensively Examined
                  • 6.3.3. New Genes Inserted into Eukaryotic Cells Can Be Efficiently Expressed
                  • 6.3.4. Transgenic Animals Harbor and Express Genes That Were Introduced into Their Germ Lines
                  • 6.3.5. Gene Disruption Provides Clues to Gene Function
                  • 6.3.6. Tumor-Inducing Plasmids Can Be Used to Introduce New Genes into Plant Cells
                  • 6.4.1. Proteins with New Functions Can Be Created Through Directed Changes in DNA
                  • 6.4.2. Recombinant DNA Technology Has Opened New Vistas
                  • The Basic Tools of Gene Exploration
                  • Recombinant DNA Technology Has Revolutionized All Aspects of Biology
                  • Manipulating the Genes of Eukaryotes
                  • Novel Proteins Can Be Engineered by Site-Specific Mutagenesis
                  • Key Terms
                  • Chapter Integration Problem
                  • Chapter Integration and Data Analysis Problem
                  • Data Interpretation Problem
                  • Where to start
                  • Books on recombinant DNA technology
                  • DNA sequencing and synthesis
                  • Polymerase chain reaction (PCR)
                  • DNA arrays
                  • Introduction of genes into animal cells
                  • Genetic engineering of plants
                  • 7.1. Homologs Are Descended from a Common Ancestor
                  • 7.2. Statistical Analysis of Sequence Alignments Can Detect Homology
                    • 7.2.1. The Statistical Significance of Alignments Can Be Estimated by Shuffling
                    • 7.2.2. Distant Evolutionary Relationships Can Be Detected Through the Use of Substitution Matrices
                    • 7.2.3. Databases Can Be Searched to Identify Homologous Sequences
                    • 7.3.1. Tertiary Structure Is More Conserved Than Primary Structure
                    • 7.3.2. Knowledge of Three-Dimensional Structures Can Aid in the Evaluation of Sequence Alignments
                    • 7.3.3. Repeated Motifs Can Be Detected by Aligning Sequences with Themselves
                    • 7.3.4. Convergent Evolution: Common Solutions to Biochemical Challenges
                    • 7.3.5. Comparison of RNA Sequences Can Be a Source of Insight into Secondary Structures
                    • 7.5.1. Ancient DNA Can Sometimes Be Amplified and Sequenced
                    • 7.5.2. Molecular Evolution Can Be Examined Experimentally
                    • Homologs Are Descended from a Common Ancestor
                    • Statistical Analysis of Sequence Alignments Can Detect Homology
                    • Examination of Three-Dimensional Structure Enhances Our Understanding of Evolutionary Relationships
                    • Evolutionary Trees Can Be Constructed on the Basis of Sequence Information
                    • Modern Techniques Make the Experimental Exploration of Evolution Possible
                    • Key Terms
                    • Media Problem
                    • Book
                    • Sequence alignment
                    • Structure comparison
                    • Domain detection
                    • Evolutionary trees
                    • Ancient DNA
                    • Evolution in the laboratory
                    • Web sites
                    • 8.1. Enzymes Are Powerful and Highly Specific Catalysts
                      • 8.1.1. Many Enzymes Require Cofactors for Activity
                      • 8.1.2. Enzymes May Transform Energy from One Form into Another
                      • 8.1.3. Enzymes Are Classified on the Basis of the Types of Reactions That They Catalyze
                      • 8.2.1. The Free-Energy Change Provides Information About the Spontaneity but Not the Rate of a Reaction
                      • 8.2.2. The Standard Free-Energy Change of a Reaction Is Related to the Equilibrium Constant
                      • 8.2.3. Enzymes Alter Only the Reaction Rate and Not the Reaction Equilibrium
                      • 8.3.1. The Formation of an Enzyme-Substrate Complex Is the First Step in Enzymatic Catalysis
                      • 8.3.2. The Active Sites of Enzymes Have Some Common Features
                      • 8.4.1. The Significance of KM and Vmax Values
                      • 8.4.2. Kinetic Perfection in Enzymatic Catalysis: The kcat/KM Criterion
                      • 8.4.3. Most Biochemical Reactions Include Multiple Substrates
                      • 8.4.4. Allosteric Enzymes Do Not Obey Michaelis-Menten Kinetics
                      • 8.5.1. Competitive and Noncompetitive Inhibition Are Kinetically Distinguishable
                      • 8.5.2. Irreversible Inhibitors Can Be Used to Map the Active Site
                      • 8.5.3. Transition-State Analogs Are Potent Inhibitors of Enzymes
                      • 8.5.4. Catalytic Antibodies Demonstrate the Importance of Selective Binding of the Transition State to Enzymatic Activity
                      • 8.5.5. Penicillin Irreversibly Inactivates a Key Enzyme in Bacterial Cell-Wall Synthesis
                      • 8.6.1. Water-Soluble Vitamins Function As Coenzymes
                      • 8.6.2. Fat-Soluble Vitamins Participate in Diverse Processes Such as Blood Clotting and Vision
                      • Enzymes are Powerful and Highly Specific Catalysts
                      • Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes
                      • Enzymes Accelerate Reactions by Facilitating the Formation of the Transition State
                      • The Michaelis-Menten Model Accounts for the Kinetic Properties of Many Enzymes
                      • Enzymes Can Be Inhibited by Specific Molecules
                      • Vitamins Are Often Precursors to Coenzymes
                      • Key Terms
                      • Data Interpretation Problems
                      • Chapter Integration Problems
                      • Media Problem
                      • Where to start
                      • Books
                      • Transition-state stabilization, analogs, and other enzyme inhibitors
                      • Catalytic antibodies
                      • Enzyme kinetics and mechanisms
                      • 9.1. Proteases: Facilitating a Difficult Reaction
                        • 9.1.1. Chymotrypsin Possesses a Highly Reactive Serine Residue
                        • 9.1.2. Chymotrypsin Action Proceeds in Two Steps Linked by a Covalently Bound Intermediate
                        • 9.1.3. Serine is Part of a Catalytic Triad That Also Includes Histidine and Aspartic Acid
                        • 9.1.4. Catalytic Triads Are Found in Other Hydrolytic Enzymes
                        • 9.1.5. The Catalytic Triad Has Been Dissected by Site-Directed Mutagenesis
                        • 9.1.6. Cysteine, Aspartyl, and Metalloproteases Are Other Major Classes of Peptide-Cleaving Enzymes
                        • 9.1.7. Protease Inhibitors Are Important Drugs
                        • 9.2.1. Carbonic Anhydrase Contains a Bound Zinc Ion Essential for Catalytic Activity
                        • 9.2.2. Catalysis Entails Zinc Activation of Water
                        • 9.2.3. A Proton Shuttle Facilitates Rapid Regeneration of the Active Form of the Enzyme
                        • 9.2.4. Convergent Evolution Has Generated Zinc-Based Active Sites in Different Carbonic Anhydrases
                        • 9.3.1. Cleavage Is by In-Line Displacement of 3′ Oxygen from Phosphorus by Magnesium-Activated Water
                        • 9.3.2. Restriction Enzymes Require Magnesium for Catalytic Activity
                        • 9.3.3. The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity
                        • 9.3.4. Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer
                        • 9.4.1. NMP Kinases Are a Family of Enzymes Containing P-Loop Structures
                        • 9.4.2. Magnesium (or Manganese) Complexes of Nucleoside Triphosphates Are the True Substrates for Essentially All NTP-Dependent Enzymes
                        • 9.4.3. ATP Binding Induces Large Conformational Changes
                        • 9.4.4. P-Loop NTPase Domains Are Present in a Range of Important Proteins
                        • Proteases: Facilitating a Difficult Reaction
                        • Carbonic Anhydrases: Making a Fast Reaction Faster
                        • Restriction Enzymes: Performing Highly Specific DNA Cleavage Reactions
                        • Nucleoside Monophosphate Kinases: Catalyzing Phosphoryl Group Exchange Without Promoting Hydrolysis
                        • Key Terms
                        • Mechanism Problem
                        • Media Problems
                        • Where to start
                        • Books
                        • Chymotrypsin and other serine proteases
                        • Other proteases
                        • Carbonic anhydrase
                        • Restriction enzymes
                        • NMP kinases
                        • 10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
                          • 10.1.1. ACTase Consists of Separable Catalytic and Regulatory Subunits
                          • 10.1.2. Allosteric Interactions in ATCase Are Mediated by Large Changes in Quaternary Structure
                          • 10.1.3. Allosterically Regulated Enzymes Do Not Follow Michaelis-Menten Kinetics
                          • 10.1.4. Allosteric Regulators Modulate the T-to-R Equilibrium
                          • 10.1.5. The Concerted Model Can Be Formulated in Quantitative Terms
                          • 10.1.6. Sequential Models Also Can Account for Allosteric Effects
                          • 10.2.1. Oxygen Binding Induces Substantial Structural Changes at the Iron Sites in Hemoglobin
                          • 10.2.2. Oxygen Binding Markedly Changes the Quaternary Structure of Hemoglobin
                          • 10.2.3. Tuning the Oxygen Affinity of Hemoglobin: The Effect of 2,3-Bisphosphoglycerate
                          • 10.2.4. The Bohr Effect: Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen
                          • 10.4.1. Phosphorylation Is a Highly Effective Means of Regulating the Activities of Target Proteins
                          • 10.4.2. Cyclic AMP Activates Protein Kinase A by Altering the Quaternary Structure
                          • 10.4.3. ATP and the Target Protein Bind to a Deep Cleft in the Catalytic Subunit of Protein Kinase A
                          • 10.5.1. Chymotrypsinogen Is Activated by Specific Cleavage of a Single Peptide Bond
                          • 10.5.2. Proteolytic Activation of Chymotrypsinogen Leads to the Formation of a Substrate-Binding Site
                          • 10.5.3. The Generation of Trypsin from Trypsinogen Leads to the Activation of Other Zymogens
                          • 10.5.4. Some Proteolytic Enzymes Have Specific Inhibitors
                          • 10.5.5. Blood Clotting Is Accomplished by a Cascade of Zymogen Activations
                          • 10.5.6. Fibrinogen Is Converted by Thrombin into a Fibrin Clot
                          • 10.5.7. Prothrombin Is Readied for Activation by a Vitamin K-Dependent Modification
                          • 10.5.8. Hemophilia Revealed an Early Step in Clotting
                          • 10.5.9. The Clotting Process Must Be Precisely Regulated
                          • Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
                          • Hemoglobin Transports Oxygen Efficiently by Binding Oxygen Cooperatively
                          • Isozymes Provide a Means of Regulation Specific to Distinct Tissues and Developmental Stages
                          • Covalent Modification Is a Means of Regulating Enzyme Activity
                          • Many Enzymes Are Activated by Specific Proteolytic Cleavage
                          • Key Terms
                          • Data Interpretation Problems
                          • Chapter Integration Problem
                          • Mechanism Problems
                          • Media Problem
                          • Where to start
                          • Aspartate transcarbamoylase and allosteric interactions
                          • Hemoglobin
                          • Covalent modification
                          • Protein kinase A
                          • Zymogen activation
                          • Protease inhibitors
                          • Clotting cascade
                          • 11.1. Monosaccharides Are Aldehydes or Ketones with Multiple Hydroxyl Groups
                            • 11.1.1. Pentoses and Hexoses Cyclize to Form Furanose and Pyranose Rings
                            • 11.1.2. Conformation of Pyranose and Furanose Rings
                            • 11.1.3. Monosaccharides Are Joined to Alcohols and Amines Through Glycosidic Bonds
                            • 11.2.1. Sucrose, Lactose, and Maltose Are the Common Disaccharides
                            • 11.2.2. Glycogen and Starch Are Mobilizable Stores of Glucose
                            • 11.2.3. Cellulose, the Major Structural Polymer of Plants, Consists of Linear Chains of Glucose Units
                            • 11.2.4. Glycosaminoglycans Are Anionic Polysaccharide Chains Made of Repeating Disaccharide Units
                            • 11.2.5. Specific Enzymes Are Responsible for Oligosaccharide Assembly
                            • 11.3.1. Carbohydrates May Be Linked to Proteins Through Asparagine (N-Linked) or Through Serine or Threonine (O-Linked) Residues
                            • 11.3.2. Protein Glycosylation Takes Place in the Lumen of the Endoplasmic Reticulum and in the Golgi Complex
                            • 11.3.3. N-Linked Glycoproteins Acquire Their Initial Sugars from Dolichol Donors in the Endoplasmic Reticulum
                            • 11.3.4. Transport Vesicles Carry Proteins from the Endoplasmic Reticulum to the Golgi Complex for Further Glycosylation and Sorting
                            • 11.3.5. Mannose 6-phosphate Targets Lysosomal Enzymes to Their Destinations
                            • 11.3.6. Glucose Residues Are Added and Trimmed to Aid in Protein Folding
                            • 11.3.7. Oligosaccharides Can Be “Sequenced”
                            • 11.4.1. Lectins Promote Interactions Between Cells
                            • 11.4.2. Influenza Virus Binds to Sialic Acid Residues
                            • Monosaccharides Are Aldehydes or Ketones with Multiple Hydroxyl Groups
                            • Complex Carbohydrates Are Formed by Linkage of Monosaccharides
                            • Carbohydrates Can Attach to Proteins to Form Glycoproteins
                            • Lectins Are Specific Carbohydrate-Binding Proteins
                            • Key Terms
                            • Chapter Integration Problem
                            • Where to start
                            • Books
                            • Structure of carbohydrate-binding proteins
                            • Glycoproteins
                            • Carbohydrates in recognition processes
                            • Carbohydrate sequencing
                            • 12.1. Many Common Features Underlie the Diversity of Biological Membranes
                            • 12.2. Fatty Acids Are Key Constituents of Lipids
                              • 12.2.1. The Naming of Fatty Acids
                              • 12.2.2. Fatty Acids Vary in Chain Length and Degree of Unsaturation
                              • 12.3.1. Phospholipids Are the Major Class of Membrane Lipids
                              • 12.3.2. Archaeal Membranes Are Built from Ether Lipids with Branched Chains
                              • 12.3.3. Membrane Lipids Can Also Include Carbohydrate Moieties
                              • 12.3.4. Cholesterol Is a Lipid Based on a Steroid Nucleus
                              • 12.3.5. A Membrane Lipid Is an Amphipathic Molecule Containing a Hydrophilic and a Hydrophobic Moiety
                              • 12.4.1. Lipid Vesicles Can Be Formed from Phospholipids
                              • 12.4.2. Lipid Bilayers Are Highly Impermeable to Ions and Most Polar Molecules
                              • 12.5.1. Proteins Associate with the Lipid Bilayer in a Variety of Ways
                              • 12.5.2. Proteins Interact with Membranes in a Variety of Ways
                              • 12.5.3. Some Proteins Associate with Membranes Through Covalently Attached Hydrophobic Groups
                              • 12.5.4. Transmembrane Helices Can Be Accurately Predicted from Amino Acid Sequences
                              • 12.6.1. The Fluid Mosaic Model Allows Lateral Movement but Not Rotation Through the Membrane
                              • 12.6.2. Membrane Fluidity Is Controlled by Fatty Acid Composition and Cholesterol Content
                              • 12.6.3. All Biological Membranes Are Asymmetric
                              • 12.7.1. Proteins Are Targeted to Specific Compartments by Signal Sequences
                              • 12.7.2. Membrane Budding and Fusion Underlie Several Important Biological Processes
                              • Many Common Features Underlie the Diversity of Biological Membranes
                              • Fatty Acids Are Key Constituents of Lipids
                              • There Are Three Common Types of Membrane Lipids
                              • Phospholipids and Glycolipids Readily Form Bimolecular Sheets in Aqueous Media
                              • Proteins Carry Out Most Membrane Processes
                              • Lipids and Many Membrane Proteins Diffuse Rapidly in the Plane of the Membrane
                              • Eukaryotic Cells Contain Compartments Bounded by Internal Membranes
                              • Key Terms
                              • Data Interpretation Problems
                              • Chapter Integration Problem
                              • Where to start
                              • Books
                              • Membrane lipids and dynamics
                              • Structure of membrane proteins
                              • Intracellular membranes
                              • 13.1. The Transport of Molecules Across a Membrane May Be Active or Passive
                                • 13.1.1. Many Molecules Require Protein Transporters to Cross Membranes
                                • 13.1.2. Free Energy Stored in Concentration Gradients Can Be Quantified
                                • 13.2.1. The Sarcoplasmic Reticulum Ca 2+ ATPase Is an Integral Membrane Protein
                                • 13.2.2. P-Type ATPases Are Evolutionarily Conserved and Play a Wide Range of Roles
                                • 13.2.3. Digitalis Specifically Inhibits the Na + -K + Pump by Blocking Its Dephosphorylation
                                • 13.5.1. Patch-Clamp Conductance Measurements Reveal the Activities of Single Channels
                                • 13.5.2. Ion-Channel Proteins Are Built of Similar Units
                                • 13.5.3. Action Potentials Are Mediated by Transient Changes in Na + and K + Permeability
                                • 13.5.4. The Sodium Channel Is an Example of a Voltage-Gated Channel
                                • 13.5.5. Potassium Channels Are Homologous to the Sodium Channel
                                • 13.5.6. The Structure of a Potassium Channel Reveals the Basis of Rapid Ion Flow with Specificity
                                • 13.5.7. The Structure of the Potassium Channel Explains Its Rapid Rates of Transport
                                • 13.5.8. A Channel Can Be Inactivated by Occlusion of the Pore: The Ball-and-Chain Model
                                • The Transport of Molecules Across a Membrane May Be Active or Passive
                                • A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes
                                • Multidrug Resistance and Cystic Fibrosis Highlight a Family of Membrane Proteins with ATP-Binding Cassette Domains
                                • Secondary Transporters Use One Concentration Gradient to Power the Formation of Another
                                • Specific Channels Can Rapidly Transport Ions Across Membranes
                                • Gap Junctions Allow Ions and Small Molecules to Flow Between Communicating Cells
                                • Key Terms
                                • Chapter Integration Problem
                                • Mechanism Problem
                                • Data Interpretation Problem
                                • Media Problem
                                • Where to start
                                • Books
                                • Voltage-gated ion channels
                                • Ligand-gated ion channels
                                • ATP-driven ion pumps
                                • ATP-binding cassette (ABC) proteins
                                • Symporters and antiporters
                                • Gap junctions
                                • Chapter 14. Metabolism: Basic Concepts and Design
                                  • 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions
                                    • 14.1.1. A Thermodynamically Unfavorable Reaction Can Be Driven by a Favorable Reaction
                                    • 14.1.2. ATP Is the Universal Currency of Free Energy in Biological Systems
                                    • 14.1.3. ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactions
                                    • 14.1.4. Structural Basis of the High Phosphoryl Transfer Potential of ATP
                                    • 14.1.5. Phosphoryl Transfer Potential Is an Important Form of Cellular Energy Transformation
                                    • 14.2.1. High Phosphoryl Transfer Potential Compounds Can Couple Carbon Oxidation to ATP Synthesis
                                    • 14.2.2. Ion Gradients Across Membranes Provide an Important Form of Cellular Energy That Can Be Coupled to ATP Synthesis
                                    • 14.2.3. Stages in the Extraction of Energy from Foodstuffs
                                    • 14.3.1. Activated Carriers Exemplify the Modular Design and Economy of Metabolism
                                    • 14.3.2. Key Reactions Are Reiterated Throughout Metabolism
                                    • 14.3.3. Metabolic Processes Are Regulated in Three Principal Ways
                                    • 14.3.4. Evolution of Metabolic Pathways
                                    • Metabolism Is Composed of Many Coupled, Interconnecting Reactions
                                    • The Oxidation of Carbon Fuels Is an Important Source of Cellular Energy
                                    • Metabolic Pathways Contain Many Recurring Motifs
                                    • Key Terms
                                    • Chapter Integration Problem
                                    • Data Interpretation
                                    • Media Problem
                                    • Where to start
                                    • Books
                                    • Thermodynamics
                                    • Bioenergetics and metabolism
                                    • Regulation of metabolism
                                    • Historical aspects
                                    • 15.1. Seven-Transmembrane-Helix Receptors Change Conformation in Response to Ligand Binding and Activate G Proteins
                                      • 15.1.1. Ligand Binding to 7TM Receptors Leads to the Activation of G Proteins
                                      • 15.1.2. G Proteins Cycle Between GDP- and GTP-Bound Forms
                                      • 15.1.3. Activated G Proteins Transmit Signals by Binding to Other Proteins
                                      • 15.1.4. G Proteins Spontaneously Reset Themselves Through GTP Hydrolysis
                                      • 15.1.5. Cyclic AMP Stimulates the Phosphorylation of Many Target Proteins by Activating Protein Kinase A
                                      • 15.2.1. Inositol 1,4,5-trisphosphate Opens Channels to Release Calcium Ions from Intracellular Stores
                                      • 15.2.2. Diacylglycerol Activates Protein Kinase C, Which Phosphorylates Many Target Proteins
                                      • 15.3.1. Ionophores Allow the Visualization of Changes in Calcium Concentration
                                      • 15.3.2. Calcium Activates the Regulatory Protein Calmodulin, Which Stimulates Many Enzymes and Transporters
                                      • 15.4.1. Some Receptors Contain Tyrosine Kinase Domains Within Their Covalent Structures
                                      • 15.4.2. Ras, Another Class of Signaling G Protein
                                      • 15.5.1. Protein Kinase Inhibitors May Be Effective Anticancer Drugs
                                      • 15.5.2. Cholera and Whooping Cough Are Due to Altered G-Protein Activity
                                      • Seven-Transmembrane-Helix Receptors Change Conformation in Response to Ligand Binding and Activate G Proteins
                                      • The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
                                      • Calcium Ion Is a Ubiquitous Cytosolic Messenger
                                      • Some Receptors Dimerize in Response to Ligand Binding and Signal by Cross-Phosphorylation
                                      • Defects in Signaling Pathways Can Lead to Cancer and Other Diseases
                                      • Recurring Features of Signal-Transduction Pathways Reveal Evolutionary Relationships
                                      • Key Terms
                                      • Chapter Integration Problem
                                      • Mechanism Problem
                                      • Data Interpretation Problems
                                      • Media Problem
                                      • Where to start
                                      • G proteins and 7TM receptors
                                      • cAMP cascade
                                      • Phosphoinositide cascade
                                      • Calcium
                                      • Protein kinases, including receptor tyrosine kinases
                                      • Ras
                                      • Cancer
                                      • 16.1. Glycolysis Is an Energy-Conversion Pathway in Many Organisms
                                        • 16.1.1. Hexokinase Traps Glucose in the Cell and Begins Glycolysis
                                        • 16.1.2. The Formation of Fructose 1,6-bisphosphate from Glucose 6-phosphate
                                        • 16.1.3. The Six-Carbon Sugar Is Cleaved into Two Three-Carbon Fragments by Aldolase
                                        • 16.1.4. Triose phosphate isomerase Salvages a Three-Carbon Fragment
                                        • 16.1.5. Energy Transformation: Phosphorylation Is Coupled to the Oxidation of Glyceraldehyde 3-phosphate by a Thioester Intermediate
                                        • 16.1.6. The Formation of ATP from 1,3-Bisphosphoglycerate
                                        • 16.1.7. The Generation of Additional ATP and the Formation of Pyruvate
                                        • 16.1.8. Energy Yield in the Conversion of Glucose into Pyruvate
                                        • 16.1.9. Maintaining Redox Balance: The Diverse Fates of Pyruvate
                                        • 16.1.10. The Binding Site for NAD + Is Similar in Many Dehydrogenases
                                        • 16.1.11. The Entry of Fructose and Galactose into Glycolysis
                                        • 16.1.12. Many Adults Are Intolerant of Milk Because They Are Deficient in Lactase
                                        • 16.1.13. Galactose Is Highly Toxic If the Transferase Is Missing
                                        • 16.2.1. Phosphofructokinase Is the Key Enzyme in the Control of Glycolysis
                                        • 16.2.2. A Regulated Bifunctional Enzyme Synthesizes and Degrades Fructose 2,6 -bisphosphate
                                        • 16.2.3. Hexokinase and Pyruvate kinase Also Set the Pace of Glycolysis
                                        • 16.2.4. A Family of Transporters Enables Glucose to Enter and Leave Animal Cells
                                        • 16.2.5. Cancer and Glycolysis
                                        • 16.3.1. Gluconeogenesis Is Not a Reversal of Glycolysis
                                        • 16.3.2. The Conversion of Pyruvate into Phosphoenolpyruvate Begins with the Formation of Oxaloacetate
                                        • 16.3.3. Oxaloacetate Is Shuttled into the Cytosol and Converted into Phosphoenolpyruvate
                                        • 16.3.4. The Conversion of Fructose 1,6-bisphosphate into Fructose 6-phosphate and Orthophosphate Is an Irreversible Step
                                        • 16.3.5. The Generation of Free Glucose Is an Important Control Point
                                        • 16.3.6. Six High Transfer Potential Phosphoryl Groups Are Spent in Synthesizing Glucose from Pyruvate
                                        • 16.4.1. Substrate Cycles Amplify Metabolic Signals and Produce Heat
                                        • 16.4.2. Lactate and Alanine Formed by Contracting Muscle Are Used by Other Organs
                                        • 16.4.3. Glycolysis and Gluconeogenesis Are Evolutionarily Intertwined
                                        • Glycolysis Is an Energy-Conversion Pathway in Many Organisms
                                        • The Glycolytic Pathway Is Tightly Controlled
                                        • Glucose Can Be Synthesized from Noncarbohydrate Precursors
                                        • Gluconeogenesis and Glycolysis Are Reciprocally Regulated
                                        • Key Terms
                                        • Mechanism Problem
                                        • Chapter Integration Problem
                                        • Data Interpretation Problem
                                        • Media Problems
                                        • Where to start
                                        • Books
                                        • Structure of glycolytic and gluconeogenic enzymes
                                        • Catalytic mechanisms
                                        • Regulation
                                        • Sugar transporters
                                        • Genetic diseases
                                        • Evolution
                                        • Historical aspects
                                        • 17.1. The Citric Acid Cycle Oxidizes Two-Carbon Units
                                          • 17.1.1. The Formation of Acetyl Coenzyme A from Pyruvate
                                          • 17.1.2. Flexible Linkages Allow Lipoamide to Move Between Different Active Sites
                                          • 17.1.3. Citrate Synthase Forms Citrate from Oxaloacetate and Acetyl Coenzyme A
                                          • 17.1.4. Citrate Is Isomerized into Isocitrate
                                          • 17.1.5. Isocitrate Is Oxidized and Decarboxylated to α-Ketoglutarate
                                          • 17.1.6. Succinyl Coenzyme A Is Formed by the Oxidative Decarboxylation of α-Ketoglutarate
                                          • 17.1.7. A High Phosphoryl-Transfer Potential Compound Is Generated from Succinyl Coenzyme A
                                          • 17.1.8. Oxaloacetate Is Regenerated by the Oxidation of Succinate
                                          • 17.1.9. Stoichiometry of the Citric Acid Cycle
                                          • 17.2.1. The Pyruvate Dehydrogenase Complex Is Regulated Allosterically and by Reversible Phosphorylation
                                          • 17.2.2. The Citric Acid Cycle Is Controlled at Several Points
                                          • 17.3.1. The Citric Acid Cycle Must Be Capable of Being Rapidly Replenished
                                          • 17.3.2. The Disruption of Pyruvate Metabolism Is the Cause of Beriberi and Poisoning by Mercury and Arsenic
                                          • 17.3.3. Speculations on the Evolutionary History of the Citric Acid Cycle
                                          • The Citric Acid Cycle Oxidizes Two-Carbon Units
                                          • Entry to the Citric Acid Cycle and Metabolism Through It Are Controlled
                                          • The Citric Acid Cycle Is a Source of Biosynthetic Precursors
                                          • The Glyoxylate Cycle Enables Plants and Bacteria to Grow on Acetate
                                          • Key Terms
                                          • Chapter Integration Problem
                                          • Mechanism Problems
                                          • Data Interpretation
                                          • Where to start
                                          • Pyruvate dehydrogenase complex
                                          • Structure of citric acid cycle enzymes
                                          • Organization of the citric acid cycle
                                          • Regulation
                                          • Evolutionary aspects
                                          • Discovery of the citric acid cycle
                                          • 18.1. Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria
                                            • 18.1.1. Mitochondria Are Bounded by a Double Membrane
                                            • 18.1.2. Mitochondria Are the Result of an Endosymbiotic Event
                                            • 18.2.1. High-Energy Electrons: Redox Potentials and Free-Energy Changes
                                            • 18.2.2. A 1.14-Volt Potential Difference Between NADH and O2 Drives Electron Transport Through the Chain and Favors the Formation of a Proton Gradient
                                            • 18.2.3. Electrons Can Be Transferred Between Groups That Are Not in Contact
                                            • 18.3.1. The High-Potential Electrons of NADH Enter the Respiratory Chain at NADH-Q Oxidoreductase
                                            • 18.3.2. Ubiquinol Is the Entry Point for Electrons from FADH2 of Flavoproteins
                                            • 18.3.3. Electrons Flow from Ubiquinol to Cytochrome c Through Q-Cytochrome c Oxidoreductase
                                            • 18.3.4. Transmembrane Proton Transport: The Q Cycle
                                            • 18.3.5. Cytochrome c Oxidase Catalyzes the Reduction of Molecular Oxygen to Water
                                            • 18.3.6. Toxic Derivatives of Molecular Oxygen Such as Superoxide Radical Are Scavenged by Protective Enzymes
                                            • 18.3.7. The Conformation of Cytochrome c Has Remained Essentially Constant for More Than a Billion Years
                                            • 18.4.1. ATP Synthase Is Composed of a Proton-Conducting Unit and a Catalytic Unit
                                            • 18.4.2. Proton Flow Through ATP Synthase Leads to the Release of Tightly Bound ATP: The Binding-Change Mechanism
                                            • 18.4.3. The World's Smallest Molecular Motor: Rotational Catalysis
                                            • 18.4.4. Proton Flow Around the c Ring Powers ATP Synthesis
                                            • 18.4.5. ATP Synthase and G Proteins Have Several Common Features
                                            • 18.5.1. Electrons from Cytosolic NADH Enter Mitochondria by Shuttles
                                            • 18.5.2. The Entry of ADP into Mitochondria Is Coupled to the Exit of ATP by ATP-ADP Translocase
                                            • 18.5.3. Mitochondrial Transporters for Metabolites Have a Common Tripartite Motif
                                            • 18.6.1. The Complete Oxidation of Glucose Yields About 30 Molecules of ATP
                                            • 18.6.2. The Rate of Oxidative Phosphorylation Is Determined by the Need for ATP
                                            • 18.6.3. Oxidative Phosphorylation Can Be Inhibited at Many Stages
                                            • 18.6.4. Regulated Uncoupling Leads to the Generation of Heat
                                            • 18.6.5. Mitochondrial Diseases Are Being Discovered
                                            • 18.6.6. Mitochondria Play a Key Role in Apoptosis
                                            • 18.6.7. Power Transmission by Proton Gradients: A Central Motif of Bioenergetics
                                            • Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria
                                            • Oxidative Phosphorylation Depends on Electron Transfer
                                            • The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle
                                            • A Proton Gradient Powers the Synthesis of ATP
                                            • Many Shuttles Allow Movement Across the Mitochondrial Membranes
                                            • The Regulation of Oxidative Phosphorylation Is Governed Primarily by the Need for ATP
                                            • Key Terms
                                            • Chapter Integration Problem
                                            • Data Interpretation Problem
                                            • Mechanism Problem
                                            • Media Problem
                                            • Where to start
                                            • Books
                                            • Electron-transport chain
                                            • ATP synthase
                                            • Translocators
                                            • Superoxide dismutase and catalase
                                            • Mitochondrial diseases
                                            • Apoptosis
                                            • Historical aspects
                                            • 19.1. Photosynthesis Takes Place in Chloroplasts
                                              • 19.1.1. The Primary Events of Photosynthesis Take Place in Thylakoid Membranes
                                              • 19.1.2. The Evolution of Chloroplasts
                                              • 19.2.1. Photosynthetic Bacteria and the Photosynthetic Reaction Centers of Green Plants Have a Common Core
                                              • 19.2.2. A Special Pair of Chlorophylls Initiates Charge Separation
                                              • 19.3.1. Photosystem II Transfers Electrons from Water to Plastoquinone and Generates a Proton Gradient
                                              • 19.3.2. Cytochrome bf Links Photosystem II to Photosystem I
                                              • 19.3.3. Photosystem I Uses Light Energy to Generate Reduced Ferredoxin, a Powerful Reductant
                                              • 19.3.4. Ferredoxin-NADP + Reductase Converts NADP + into NADPH
                                              • 19.4.1. The ATP Synthase of Chloroplasts Closely Resembles Those of Mitochondria and Prokaryotes
                                              • 19.4.2. Cyclic Electron Flow Through Photosystem I Leads to the Production of ATP Instead of NADPH
                                              • 19.4.3. The Absorption of Eight Photons Yields One O2, Two NADPH, and Three ATP Molecules
                                              • 19.5.1. Resonance Energy Transfer Allows Energy to Move from the Site of Initial Absorbance to the Reaction Center
                                              • 19.5.2. Light-Harvesting Complexes Contain Additional Chlorophylls and Carotinoids
                                              • 19.5.3. Phycobilisomes Serve as Molecular Light Pipes in Cyanobacteria and Red Algae
                                              • 19.5.4. Components of Photosynthesis Are Highly Organized
                                              • 19.5.5. Many Herbicides Inhibit the Light Reactions of Photosynthesis
                                              • Photosynthesis Takes Place in Chloroplasts
                                              • Light Absorption by Chlorophyll Induces Electron Transfer
                                              • Two Photosystems Generate a Proton Gradient and NADPH in Oxygenic Photosynthesis
                                              • A Proton Gradient Across the Thylakoid Membrane Drives ATP Synthesis
                                              • Accessory Pigments Funnel Energy into Reaction Centers
                                              • The Ability to Convert Light into Chemical Energy Is Ancient
                                              • Key Terms
                                              • Mechanism Problem
                                              • Data Interpretation and Chapter Integration Problem
                                              • Where to start
                                              • Books and general reviews
                                              • Electron-transfer mechanisms
                                              • Photosystem II
                                              • Oxygen evolution
                                              • Photosystem I and cytochrome bf
                                              • ATP synthase
                                              • Light-harvesting assemblies
                                              • Evolution
                                              • 20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
                                                • 20.1.1. Carbon Dioxide Reacts with Ribulose 1,5-bisphosphate to Form Two Molecules of 3-Phosphoglycerate
                                                • 20.1.2. Catalytic Imperfection: Rubisco Also Catalyzes a Wasteful Oxygenase Reaction
                                                • 20.1.3. Hexose Phosphates Are Made from Phosphoglycerate, and Ribulose 1,5-bisphosphate Is Regenerated
                                                • 20.1.4. Three Molecules of ATP and Two Molecules of NADPH Are Used to Bring Carbon Dioxide to the Level of a Hexose
                                                • 20.1.5. Starch and Sucrose Are the Major Carbohydrate Stores in Plants
                                                • 20.2.1. Rubisco Is Activated by Light-Driven Changes in Proton and Magnesium Ion Concentrations
                                                • 20.2.2. Thioredoxin Plays a Key Role in Regulating the Calvin Cycle
                                                • 20.2.3. The C4 Pathway of Tropical Plants Accelerates Photosynthesis by Concentrating Carbon Dioxide
                                                • 20.2.4. Crassulacean Acid Metabolism Permits Growth in Arid Ecosystems
                                                • 20.3.1. Two Molecules of NADPH Are Generated in the Conversion of Glucose 6-phosphate into Ribulose 5-phosphate
                                                • 20.3.2. The Pentose Phosphate Pathway and Glycolysis Are Linked by Transketolase and Transaldolase
                                                • 20.3.3. Transketolase and Transaldolase Stabilize Carbanionic Intermediates by Different Mechanisms
                                                • 20.4.1. The Rate of the Pentose Phosphate Pathway Is Controlled by the Level of NADP +
                                                • 20.4.2. The Flow of Glucose 6-phosphate Depends on the Need for NADPH, Ribose 5-phosphate, and ATP
                                                • 20.4.3. Through the Looking Glass: The Calvin Cycle and the Pentose Phosphate Pathway
                                                • 20.5.1. Glucose 6-phosphate Dehydrogenase Deficiency Causes a Drug-Induced Hemolytic Anemia
                                                • 20.5.2. A Deficiency of Glucose 6-phosphate Dehydrogenase Confers an Evolutionary Advantage in Some Circumstances
                                                • The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
                                                • The Activity of the Calvin Cycle Depends on Environmental Conditions
                                                • The Pentose Phosphate Pathway Generates NADPH and Synthesizes Five-Carbon Sugars
                                                • The Metabolism of Glucose 6-phosphate by the Pentose Phosphate Pathway Is Coordinated with Glycolysis
                                                • Glucose 6-phosphate Dehydrogenase Plays a Key Role in Protection Against Reactive Oxygen Species
                                                • Key Terms
                                                • Mechanism Problems
                                                • Chapter Integration Problems
                                                • Date Interpretation Problem
                                                • Where to start
                                                • Books and general reviews
                                                • Enzymes and reaction mechanisms
                                                • Carbon dioxide fixation and rubisco
                                                • Regulation
                                                • Glucose 6-phosphate dehydrogenase
                                                • Evolution
                                                • 21.1. Glycogen Breakdown Requires the Interplay of Several Enzymes
                                                  • 21.1.1. Phosphorylase Catalyzes the Phosphorolytic Cleavage of Glycogen to Release Glucose 1-phosphate
                                                  • 21.1.2. A Debranching Enzyme Also Is Needed for the Breakdown of Glycogen
                                                  • 21.1.3. Phosphoglucomutase Converts Glucose 1-phosphate into Glucose 6-phosphate
                                                  • 21.1.4. Liver Contains Glucose 6-phosphatase, a Hydrolytic Enzyme Absent from Muscle
                                                  • 21.1.5. Pyridoxal Phosphate Participates in the Phosphorolytic Cleavage of Glycogen
                                                  • 21.2.1. Muscle Phosphorylase Is Regulated by the Intracellular Energy Charge
                                                  • 21.2.2. Liver Phosphorylase Produces Glucose for Use by Other Tissues
                                                  • 21.2.3. Phosphorylase Kinase Is Activated by Phosphorylation and Calcium Ions
                                                  • 21.3.1. G Proteins Transmit the Signal for the Initiation of Glycogen Breakdown
                                                  • 21.3.2. Glycogen Breakdown Must Be Capable of Being Rapidly Turned Off
                                                  • 21.3.3. The Regulation of Glycogen Phosphorylase Became More Sophisticated as the Enzyme Evolved
                                                  • 21.4.1. UDP-Glucose Is an Activated Form of Glucose
                                                  • 21.4.2. Glycogen Synthase Catalyzes the Transfer of Glucose from UDP-Glucose to a Growing Chain
                                                  • 21.4.3. A Branching Enzyme Forms α-1,6 Linkages
                                                  • 21.4.4. Glycogen Synthase Is the Key Regulatory Enzyme in Glycogen Synthesis
                                                  • 21.4.5. Glycogen Is an Efficient Storage Form of Glucose
                                                  • 21.5.1. Protein Phosphatase 1 Reverses the Regulatory Effects of Kinases on Glycogen Metabolism
                                                  • 21.5.2. Insulin Stimulates Glycogen Synthesis by Activating Protein Phosphatase 1
                                                  • 21.5.3. Glycogen Metabolism in the Liver Regulates the Blood-Glucose Level
                                                  • 21.5.4. A Biochemical Understanding of Glycogen-Storage Diseases Is Possible
                                                  • Glycogen Breakdown Requires the Interplay of Several Enzymes
                                                  • Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation
                                                  • Epinephrine and Glucagon Signal the Need for Glycogen Breakdown
                                                  • Glycogen Is Synthesized and Degraded by Different Pathways
                                                  • Glycogen Breakdown and Synthesis Are Reciprocally Regulated
                                                  • Key Terms
                                                  • Mechanism Problem
                                                  • Chapter Integration and Data Interpretation Problems
                                                  • Media Problem
                                                  • Where to start
                                                  • Books and general reviews
                                                  • X-ray crystallographic studies
                                                  • Priming of glycogen synthesis
                                                  • Catalytic mechanisms
                                                  • Regulation of glycogen metabolism
                                                  • Genetic diseases
                                                  • Evolution
                                                  • 22.1. Triacylglycerols Are Highly Concentrated Energy Stores
                                                    • 22.1.1. Dietary Lipids Are Digested by Pancreatic Lipases
                                                    • 22.1.2. Dietary Lipids Are Transported in Chylomicrons
                                                    • 22.2.1. Triacylglycerols Are Hydrolyzed by Cyclic AMP-Regulated Lipases
                                                    • 22.2.2. Fatty Acids Are Linked to Coenzyme A Before They Are Oxidized
                                                    • 22.2.3. Carnitine Carries Long-Chain Activated Fatty Acids into the Mitochondrial Matrix
                                                    • 22.2.4. Acetyl CoA, NADH, and FADH2 Are Generated in Each Round of Fatty Acid Oxidation
                                                    • 22.2.5. The Complete Oxidation of Palmitate Yields 106 Molecules of ATP
                                                    • 22.3.1. An Isomerase and a Reductase Are Required for the Oxidation of Unsaturated Fatty Acids
                                                    • 22.3.2. Odd-Chain Fatty Acids Yield Propionyl Coenzyme A in the Final Thiolysis Step
                                                    • 22.3.3. Propionyl CoA Is Converted into Succinyl CoA in a Reaction That Requires Vitamin B12
                                                    • 22.3.4. Fatty Acids Are Also Oxidized in Peroxisomes
                                                    • 22.3.5. Ketone Bodies Are Formed from Acetyl Coenzyme A When Fat Breakdown Predominates
                                                    • 22.3.6. Ketone Bodies Are a Major Fuel in Some Tissues
                                                    • 22.3.7. Animals Cannot Convert Fatty Acids into Glucose
                                                    • 22.4.1. The Formation of Malonyl Coenzyme A Is the Committed Step in Fatty Acid Synthesis
                                                    • 22.4.2. Intermediates in Fatty Acid Synthesis Are Attached to an Acyl Carrier Protein
                                                    • 22.4.3. The Elongation Cycle in Fatty Acid Synthesis
                                                    • 22.4.4. Fatty Acids Are Synthesized by a Multifunctional Enzyme Complex in Eukaryotes
                                                    • 22.4.5. The Flexible Phosphopantetheinyl Unit of ACP Carries Substrate from One Active Site to Another
                                                    • 22.4.6. The Stoichiometry of Fatty Acid Synthesis
                                                    • 22.4.7. Citrate Carries Acetyl Groups from Mitochondria to the Cytosol for Fatty Acid Synthesis
                                                    • 22.4.8. Sources of NADPH for Fatty Acid Synthesis
                                                    • 22.4.9. Fatty Acid Synthase Inhibitors May Be Useful Drugs
                                                    • 22.4.10. Variations on a Theme: Polyketide and Nonribosomal Peptide Synthetases Resemble Fatty Acid Synthase
                                                    • Global Regulation
                                                    • Local Regulation
                                                    • Response to Diet
                                                    • 22.6.1. Membrane-Bound Enzymes Generate Unsaturated Fatty Acids
                                                    • 22.6.2. Eicosanoid Hormones Are Derived from Polyunsaturated Fatty Acids
                                                    • Triacylglycerols Are Highly Concentrated Energy Stores
                                                    • The Utilization of Fatty Acids as Fuel Requires Three Stages of Processing
                                                    • Certain Fatty Acids Require Additional Steps for Degradation
                                                    • Fatty Acids Are Synthesized and Degraded by Different Pathways
                                                    • Acetyl Coenzyme A Carboxylase Plays a Key Role in Controlling Fatty Acid Metabolism
                                                    • Elongation and Unsaturation of Fatty Acids Are Accomplished by Accessory Enzyme Systems
                                                    • Key Terms
                                                    • Mechanism Problems
                                                    • Chapter Integration Problems
                                                    • Data Interpretation Problem
                                                    • Media Problem
                                                    • Where to start
                                                    • Books
                                                    • Fatty acid oxidation
                                                    • Fatty acid synthesis
                                                    • Acetyl CoA carboxylase
                                                    • Eicosanoids
                                                    • Genetic diseases
                                                    • 23.1. Proteins Are Degraded to Amino Acids
                                                      • 23.1.1. The Digestion and Absorption of Dietary Proteins
                                                      • 23.1.2. Cellular Proteins Are Degraded at Different Rates
                                                      • 23.2.1. Ubiquitin Tags Proteins for Destruction
                                                      • 23.2.2. The Proteasome Digests the Ubiquitin-Tagged Proteins
                                                      • 23.2.3. Protein Degradation Can Be Used to Regulate Biological Function
                                                      • 23.2.4. The Ubiquitin Pathway and the Proteasome Have Prokaryotic Counterparts
                                                      • 23.3.1. Alpha-Amino Groups Are Converted into Ammonium Ions by the Oxidative Deamination of Glutamate
                                                      • 23.3.2. Pyridoxal Phosphate Forms Schiff-Base Intermediates in Aminotransferases
                                                      • 23.3.3. Aspartate Aminotransferase Is a Member of a Large and Versatile Family of Pyridoxal-Dependent Enzymes
                                                      • 23.3.4. Serine and Threonine Can Be Directly Deaminated
                                                      • 23.3.5. Peripheral Tissues Transport Nitrogen to the Liver
                                                      • 23.4.1. The Urea Cycle Begins with the Formation of Carbamoyl Phosphate
                                                      • 23.4.2. The Urea Cycle Is Linked to the Citric Acid Cycle
                                                      • 23.4.3. The Evolution of the Urea Cycle
                                                      • 23.4.4. Inherited Defects of the Urea Cycle Cause Hyperammonemia and Can Lead to Brain Damage
                                                      • 23.4.5. Urea Is Not the Only Means of Disposing of Excess Nitrogen
                                                      • 23.5.1. Pyruvate as an Entry Point into Metabolism
                                                      • 23.5.2. Oxaloacetate as an Entry Point into Metabolism
                                                      • 23.5.3. Alpha-Ketoglutarate as an Entry Point into Metabolism
                                                      • 23.5.4. Succinyl Coenzyme A Is a Point of Entry for Several Nonpolar Amino Acids
                                                      • 23.5.5. Methionine Degradation Requires the Formation of a Key Methyl Donor, S-Adenosylmethionine
                                                      • 23.5.6. The Branched-Chain Amino Acids Yield Acetyl CoA, Acetoacetate, or Propionyl CoA
                                                      • 23.5.7. Oxygenases Are Required for the Degradation of Aromatic Amino Acids
                                                      • Proteins Are Degraded to Amino Acids
                                                      • Protein Turnover Is Tightly Regulated
                                                      • The First Step in Amino Acid Degradation Is the Removal of Nitrogen
                                                      • Ammonium Ion Is Converted into Urea in Most Terrestrial Vertebrates
                                                      • Carbon Atoms of Degraded Amino Acids Emerge as Major Metabolic Intermediates
                                                      • Inborn Errors of Metabolism Can Disrupt Amino Acid Degradation
                                                      • Key Terms
                                                      • Mechanism Problems
                                                      • Chapter Integration Problems
                                                      • Data Interpretation Problem
                                                      • Where to start
                                                      • Books
                                                      • Ubiquitin and the proteasome
                                                      • Pyridoxal phosphate-dependent enzymes
                                                      • Urea-cycle enzymes
                                                      • Amino acid degradation
                                                      • Genetic diseases
                                                      • Historical aspects and the process of discovery
                                                      • Chapter 24. The Biosynthesis of Amino Acids
                                                        • 24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia
                                                          • 24.1.1. The Iron-Molybdenum Cofactor of Nitrogenase Binds and Reduces Atmospheric Nitrogen
                                                          • 24.1.2. Ammonium Ion Is Assimilated into an Amino Acid Through Glutamate and Glutamine
                                                          • 24.2.1. Human Beings Can Synthesize Some Amino Acids but Must Obtain Others from the Diet
                                                          • 24.2.2. A Common Step Determines the Chirality of All Amino Acids
                                                          • 24.2.3. An Adenylated Intermediate Is Required to Form Asparagine from Aspartate
                                                          • 24.2.4. Glutamate Is the Precursor of Glutamine, Proline, and Arginine
                                                          • 24.2.5. Serine, Cysteine, and Glycine Are Formed from 3-Phosphoglycerate
                                                          • 24.2.6. Tetrahydrofolate Carries Activated One-Carbon Units at Several Oxidation Levels
                                                          • 24.2.7. S-Adenosylmethionine Is the Major Donor of Methyl Groups
                                                          • 24.2.8. Cysteine Is Synthesized from Serine and Homocysteine
                                                          • 24.2.9. High Homocysteine Levels Are Associated with Vascular Disease
                                                          • 24.2.10. Shikimate and Chorismate Are Intermediates in the Biosynthesis of Aromatic Amino Acids
                                                          • 24.2.11. Tryptophan Synthetase Illustrates Substrate Channeling in Enzymatic Catalysis
                                                          • 24.3.1. Branched Pathways Require Sophisticated Regulation
                                                          • 24.3.2. The Activity of Glutamine Synthetase Is Modulated by an Enzymatic Cascade
                                                          • 24.4.1. Glutathione, a Gamma-Glutamyl Peptide, Serves as a Sulfhydryl Buffer and an Antioxidant
                                                          • 24.4.2. Nitric Oxide, a Short-Lived Signal Molecule, Is Formed from Arginine
                                                          • 24.4.3. Mammalian Porphyrins Are Synthesized from Glycine and Succinyl Coenzyme A
                                                          • 24.4.4. Porphyrins Accumulate in Some Inherited Disorders of Porphyrin Metabolism
                                                          • Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce Atmospheric Nitrogen to Ammonia
                                                          • Amino Acids Are Made from Intermediates of the Citric Acid Cycle and Other Major Pathways
                                                          • Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
                                                          • Amino Acids Are Precursors of Many Biomolecules
                                                          • Key Terms
                                                          • Mechanism Problems
                                                          • Chapter Integration Problems
                                                          • Chapter Integration and Data Interpretation Problem
                                                          • Where to start
                                                          • Books
                                                          • Nitrogen fixation
                                                          • Regulation of amino acid biosynthesis
                                                          • Aromatic amino acid biosynthesis
                                                          • Glutathione
                                                          • Ethylene and nitric oxide
                                                          • Biosynthesis of porphyrins
                                                          • 25.1. In de Novo Synthesis, the Pyrimidine Ring Is Assembled from Bicarbonate, Aspartate, and Glutamine
                                                            • 25.1.1. Bicarbonate and Other Oxygenated Carbon Compounds Are Activated by Phosphorylation
                                                            • 25.1.2. The Side Chain of Glutamine Can Be Hydrolyzed to Generate Ammonia
                                                            • 25.1.3. Intermediates Can Move Between Active Sites by Channeling
                                                            • 25.1.4. Orotate Acquires a Ribose Ring from PRPP to Form a Pyrimidine Nucleotide and Is Converted into Uridylate
                                                            • 25.1.5. Nucleotide Mono-, Di-, and Triphosphates Are Interconvertible
                                                            • 25.1.6. CTP Is Formed by Amination of UTP
                                                            • 25.2.1. Salvage Pathways Economize Intracellular Energy Expenditure
                                                            • 25.2.2. The Purine Ring System Is Assembled on Ribose Phosphate
                                                            • 25.2.3. The Purine Ring Is Assembled by Successive Steps of Activation by Phosphorylation Followed by Displacement
                                                            • 25.2.4. AMP and GMP Are Formed from IMP
                                                            • 25.3.1. Thymidylate Is Formed by the Methylation of Deoxyuridylate
                                                            • 25.3.2. Dihydrofolate Reductase Catalyzes the Regeneration of Tetrahydrofolate, a One-Carbon Carrier
                                                            • 25.3.3. Several Valuable Anticancer Drugs Block the Synthesis of Thymidylate
                                                            • 25.6.1. Purines Are Degraded to Urate in Human Beings
                                                            • 25.6.2. Lesch-Nyhan Syndrome Is a Dramatic Consequence of Mutations in a Salvage-Pathway Enzyme
                                                            • In de Novo Synthesis, the Pyrimidine Ring Is Assembled from Bicarbonate, Aspartate, and Glutamine
                                                            • Purines Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways
                                                            • Deoxyribonucleotides Are Synthesized by the Reduction of Ribonucleotides Through a Radical Mechanism
                                                            • Key Steps in Nucleotide Biosynthesis Are Regulated by Feedback Inhibition
                                                            • NAD + , FAD, and Coenzyme A Are Formed from ATP
                                                            • Disruptions in Nucleotide Metabolism Can Cause Pathological Conditions
                                                            • Key Terms
                                                            • Mechanism Problems
                                                            • Chapter Integration Problems
                                                            • Where to start
                                                            • Pyrimidine biosynthesis
                                                            • Purine biosynthesis
                                                            • Ribonucleotide reductases
                                                            • Thymidylate synthase and dihydrofolate reductase
                                                            • Genetic diseases
                                                            • 26.1. Phosphatidate Is a Common Intermediate in the Synthesis of Phospholipids and Triacylglycerols
                                                              • 26.1.1. The Synthesis of Phospholipids Requires an Activated Intermediate
                                                              • 26.1.2. Plasmalogens and Other Ether Phospholipids Are Synthesized from Dihydroxyacetone Phosphate
                                                              • 26.1.3. Sphingolipids Are Synthesized from Ceramide
                                                              • 26.1.4. Gangliosides Are Carbohydrate-Rich Sphingolipids That Contain Acidic Sugars
                                                              • 26.1.5. Sphingolipids Confer Diversity on Lipid Structure and Function
                                                              • 26.1.6. Respiratory Distress Syndrome and Tay-Sachs Disease Result from the Disruption of Lipid Metabolism
                                                              • 26.2.1. The Synthesis of Mevalonate, Which Is Activated as Isopentenyl Pyrophosphate, Initiates the Synthesis of Cholesterol
                                                              • 26.2.2. Squalene (C30) Is Synthesized from Six Molecules of Isopentenyl Pyrophosphate (C5)
                                                              • 26.2.3. Squalene Cyclizes to Form Cholesterol
                                                              • 26.3.1. Lipoproteins Transport Cholesterol and Triacylglycerols Throughout the Organism
                                                              • 26.3.2. The Blood Levels of Certain Lipoproteins Can Serve Diagnostic Purposes
                                                              • 26.3.3. Low-Density Lipoproteins Play a Central Role in Cholesterol Metabolism
                                                              • 26.3.4. The LDL Receptor Is a Transmembrane Protein Having Five Different Functional Regions
                                                              • 26.3.5. The Absence of the LDL Receptor Leads to Hypercholesteremia and Atherosclerosis
                                                              • 26.3.6. The Clinical Management of Cholesterol Levels Can Be Understood at a Biochemical Level
                                                              • Bile Salts
                                                              • Steroid Hormones
                                                              • 26.4.1. The Nomenclature of Steroid Hormones
                                                              • 26.4.2. Steroids Are Hydroxylated by Cytochrome P450 Monooxygenases That Utilize NADPH and O2
                                                              • 26.4.3. The Cytochrome P450 System Is Widespread and Performs a Protective Function
                                                              • 26.4.4. Pregnenolone, a Precursor for Many Other Steroids, Is Formed from Cholesterol by Cleavage of Its Side Chain
                                                              • 26.4.5. The Synthesis of Progesterone and Corticosteroids from Pregnenolone
                                                              • 26.4.6. The Synthesis of Androgens and Estrogens from Pregnenolone
                                                              • 26.4.7. Vitamin D Is Derived from Cholesterol by the Ring-Splitting Activity of Light
                                                              • 26.4.8. Isopentenyl Pyrophosphate Is a Precursor for a Wide Variety of Biomolecules
                                                              • Phosphatidate Is a Common Intermediate in the Synthesis of Phospholipids and Triacylglycerols
                                                              • Cholesterol Is Synthesized from Acetyl Coenzyme A in Three Stages
                                                              • The Complex Regulation of Cholesterol Biosynthesis Takes Place at Several Levels
                                                              • Important Derivatives of Cholesterol Include Bile Salts and Steroid Hormones
                                                              • Key Terms
                                                              • Mechanism Problem
                                                              • Data Interpretation and Chapter Integration Problems
                                                              • Where to start
                                                              • Books
                                                              • Phospholipids and sphingolipids
                                                              • Biosynthesis of cholesterol and steroids
                                                              • Lipoproteins and their receptors
                                                              • Oxygen activation and P450 catalysis
                                                              • 27.1. DNA Can Assume a Variety of Structural Forms
                                                                • 27.1.1. A-DNA Is a Double Helix with Different Characteristics from Those of the More Common B-DNA
                                                                • 27.1.2. The Major and Minor Grooves Are Lined by Sequence-Specific Hydrogen-Bonding Groups
                                                                • 27.1.3. The Results of Studies of Single Crystals of DNA Revealed Local Variations in DNA Structure
                                                                • 27.1.4. Z-DNA Is a Left-Handed Double Helix in Which Backbone Phosphates Zigzag
                                                                • 27.2.1. All DNA Polymerases Have Structural Features in Common
                                                                • 27.2.2. Two Bound Metal Ions Participate in the Polymerase Reaction
                                                                • 27.2.3. The Specificity of Replication Is Dictated by Hydrogen Bonding and the Complementarity of Shape Between Bases
                                                                • 27.2.4. Many Polymerases Proofread the Newly Added Bases and Excise Errors
                                                                • 27.2.5. The Separation of DNA Strands Requires Specific Helicases and ATP Hydrolysis
                                                                • 27.3.1. The Linking Number of DNA, a Topological Property, Determines the Degree of Supercoiling
                                                                • 27.3.2. Helical Twist and Superhelical Writhe Are Correlated with Each Other Through the Linking Number
                                                                • 27.3.3. Type I Topoisomerases Relax Supercoiled Structures
                                                                • 27.3.4. Type II Topoisomerases Can Introduce Negative Supercoils Through Coupling to ATP Hydrolysis
                                                                • 27.4.1. An RNA Primer Synthesized by Primase Enables DNA Synthesis to Begin
                                                                • 27.4.2. One Strand of DNA Is Made Continuously, Whereas the Other Strand Is Synthesized in Fragments
                                                                • 27.4.3. DNA Ligase Joins Ends of DNA in Duplex Regions
                                                                • 27.4.4. DNA Replication Requires Highly Processive Polymerases
                                                                • 27.4.5. The Leading and Lagging Strands Are Synthesized in a Coordinated Fashion
                                                                • 27.4.6. DNA Synthesis Is More Complex in Eukaryotes Than in Prokaryotes
                                                                • 27.4.7. Telomeres Are Unique Structures at the Ends of Linear Chromosomes
                                                                • 27.4.8. Telomeres Are Replicated by Telomerase, a Specialized Polymerase That Carries Its Own RNA Template
                                                                • 27.5.1. Recombination Reactions Proceed Through Holliday Junction Intermediates
                                                                • 27.5.2. Recombinases Are Evolutionarily Related to Topoisomerases
                                                                • 27.6.1. Some Chemical Mutagens Are Quite Specific
                                                                • 27.6.2. Ultraviolet Light Produces Pyrimidine Dimers
                                                                • 27.6.3. A Variety of DNA-Repair Pathways Are Utilized
                                                                • 27.6.4. The Presence of Thymine Instead of Uracil in DNA Permits the Repair of Deaminated Cytosine
                                                                • 27.6.5. Many Cancers Are Caused by Defective Repair of DNA
                                                                • 27.6.6. Some Genetic Diseases Are Caused by the Expansion of Repeats of Three Nucleotides
                                                                • 27.6.7. Many Potential Carcinogens Can Be Detected by Their Mutagenic Action on Bacteria
                                                                • DNA Can Assume a Variety of Structural Forms
                                                                • DNA Polymerases Require a Template and a Primer
                                                                • Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
                                                                • DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites
                                                                • Double-Stranded DNA Molecules with Similar Sequences Sometimes Recombine
                                                                • Mutations Are Produced by Several Types of Changes in the Base Sequence of DNA
                                                                • Key Terms
                                                                • Mechanism Problems
                                                                • Data Interpretation and Chapter Integration Problems
                                                                • Media Problem
                                                                • Where to begin
                                                                • Books
                                                                • DNA structure
                                                                • DNA topology and topoisomerases
                                                                • Mechanism of replication
                                                                • DNA polymerases and other enzymes of replication
                                                                • Recombinases
                                                                • Mutations and DNA repair
                                                                • Defective DNA repair and cancer
                                                                • 28.1. Transcription Is Catalyzed by RNA Polymerase
                                                                  • 28.1.1. Transcription Is Initiated at Promoter Sites on the DNA Template
                                                                  • 28.1.2. Sigma Subunits of RNA Polymerase Recognize Promoter Sites
                                                                  • 28.1.3. RNA Polymerase Must Unwind the Template Double Helix for Transcription to Take Place
                                                                  • 28.1.4. RNA Chains Are Formed de Novo and Grow in the 5′-to-3′ Direction
                                                                  • 28.1.5. Elongation Takes Place at Transcription Bubbles That Move Along the DNA Template
                                                                  • 28.1.6. An RNA Hairpin Followed by Several Uracil Residues Terminates the Transcription of Some Genes
                                                                  • 28.1.7. The Rho Protein Helps Terminate the Transcription of Some Genes
                                                                  • 28.1.8. Precursors of Transfer and Ribosomal RNA Are Cleaved and Chemically Modified After Transcription
                                                                  • 28.1.9. Antibiotic Inhibitors of Transcription
                                                                  • 28.2.1. RNA in Eukaryotic Cells Is Synthesized by Three Types of RNA Polymerase
                                                                  • 28.2.2. Cis- And Trans-Acting Elements: Locks and Keys of Transcription
                                                                  • 28.2.3. Most Promoters for RNA Polymerase II Contain a TATA Box Near the Transcription Start Site
                                                                  • 28.2.4. The TATA-Box-Binding Protein Initiates the Assembly of the Active Transcription Complex
                                                                  • 28.2.5. Multiple Transcription Factors Interact with Eukaryotic Promoters
                                                                  • 28.2.6. Enhancer Sequences Can Stimulate Transcription at Start Sites Thousands of Bases Away
                                                                  • 28.3.1. The Ends of the Pre-mRNA Transcript Acquire a 5′ Cap and a 3′ Poly(A) Tail
                                                                  • 28.3.2. RNA Editing Changes the Proteins Encoded by mRNA
                                                                  • 28.3.3. Splice Sites in mRNA Precursors Are Specified by Sequences at the Ends of Introns
                                                                  • 28.3.4. Splicing Consists of Two Transesterification Reactions
                                                                  • 28.3.5. Small Nuclear RNAs in Spliceosomes Catalyze the Splicing of mRNA Precursors
                                                                  • 28.3.6. Some Pre-mRNA Molecules Can Be Spliced in Alternative Ways to Yield Different mRNAs
                                                                  • Transcription Is Catalyzed by RNA Polymerase
                                                                  • Eukaryotic Transcription and Translation Are Separated in Space and Time
                                                                  • The Transcription Products of All Three Eukaryotic Polymerases Are Processed
                                                                  • The Discovery of Catalytic RNA Was Revealing with Regard to Both Mechanism And Evolution
                                                                  • Key Terms
                                                                  • Mechanism Problem
                                                                  • Chapter Integration Problems
                                                                  • Data Interpretation Problems
                                                                  • Where to begin
                                                                  • Books
                                                                  • RNA polymerases
                                                                  • Initiation and elongation
                                                                  • Promoters, enhancers, and transcription factors
                                                                  • Termination
                                                                  • 5′-Cap formation and polyadenylation
                                                                  • RNA editing
                                                                  • Splicing of mRNA precursors
                                                                  • Self-splicing and RNA catalysis
                                                                  • 29.1. Protein Synthesis Requires the Translation of Nucleotide Sequences Into Amino Acid Sequences
                                                                    • 29.1.1. The Synthesis of Long Proteins Requires a Low Error Frequency
                                                                    • 29.1.2. Transfer RNA Molecules Have a Common Design
                                                                    • 29.1.3. The Activated Amino Acid and the Anticodon of tRNA Are at Opposite Ends of the L-Shaped Molecule
                                                                    • 29.2.1. Amino Acids Are First Activated by Adenylation
                                                                    • 29.2.2. Aminoacyl-tRNA Synthetases Have Highly Discriminating Amino Acid Activation Sites
                                                                    • 29.2.3. Proofreading by Aminoacyl-tRNA Synthetases Increases the Fidelity of Protein Synthesis
                                                                    • 29.2.4. Synthetases Recognize the Anticodon Loops and Acceptor Stems of Transfer RNA Molecules
                                                                    • 29.2.5. Aminoacyl-tRNA Synthetases Can Be Divided into Two Classes
                                                                    • 29.3.1. Ribosomal RNAs (5S, 16S, and 23S rRNA) Play a Central Role in Protein Synthesis
                                                                    • 29.3.2. Proteins Are Synthesized in the Amino-to-Carboxyl Direction
                                                                    • 29.3.3. Messenger RNA Is Translated in the 5′-to-3′ Direction
                                                                    • 29.3.4. The Start Signal Is AUG (or GUG) Preceded by Several Bases That Pair with 16S rRNA
                                                                    • 29.3.5. Bacterial Protein Synthesis Is Initiated by Formylmethionyl Transfer RNA
                                                                    • 29.3.6. Ribosomes Have Three tRNA-Binding Sites That Bridge the 30S and 50S Subunits
                                                                    • 29.3.7. The Growing Polypeptide Chain Is Transferred Between tRNAs on Peptide-Bond Formation
                                                                    • 29.3.8. Only the Codon-Anticodon Interactions Determine the Amino Acid That Is Incorporated
                                                                    • 29.3.9. Some Transfer RNA Molecules Recognize More Than One Codon Because of Wobble in Base-Pairing
                                                                    • 29.4.1. Formylmethionyl-tRNAf Is Placed in the P Site of the Ribosome During Formation of the 70S Initiation Complex
                                                                    • 29.4.2. Elongation Factors Deliver Aminoacyl-tRNA to the Ribosome
                                                                    • 29.4.3. The Formation of a Peptide Bond Is Followed by the GTP-Driven Translocation of tRNAs and mRNA
                                                                    • 29.4.4. Protein Synthesis Is Terminated by Release Factors That Read Stop Codons
                                                                    • 29.5.1. Many Antibiotics Work by Inhibiting Protein Synthesis
                                                                    • 29.5.2. Diphtheria Toxin Blocks Protein Synthesis in Eukaryotes by Inhibiting Translocation
                                                                    • Protein Synthesis Requires the Translation of Nucleotide Sequences into Amino Acid Sequences
                                                                    • Aminoacyl-Transfer-RNA Synthetases Read the Genetic Code
                                                                    • A Ribosome Is a Ribonucleoprotein Particle (70S) Made of a Small (30S) and a Large (50S) Subunit
                                                                    • Protein Factors Play Key Roles in Protein Synthesis
                                                                    • Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation
                                                                    • Key Terms
                                                                    • Mechanism Problems
                                                                    • Chapter Integration Problems
                                                                    • Data Interpretation Problem
                                                                    • Media Problem
                                                                    • Where to start
                                                                    • Books
                                                                    • Aminoacyl-tRNA synthetases
                                                                    • Transfer RNA
                                                                    • Ribosomes and ribosomal RNAs
                                                                    • Initiation factors
                                                                    • Elongation factors
                                                                    • Peptide-bond formation and translocation
                                                                    • Termination
                                                                    • Fidelity and proofreading
                                                                    • Eukaryotic protein synthesis
                                                                    • Antibiotics and toxins
                                                                    • 30.1. Metabolism Consist of Highly Interconnected Pathways
                                                                      • 30.1.1. Recurring Motifs in Metabolic Regulation
                                                                      • 30.1.2. Major Metabolic Pathways and Control Sites
                                                                      • 30.1.3. Key Junctions: Glucose 6-phosphate, Pyruvate, and Acetyl CoA
                                                                      • 30.3.1. Metabolic Adaptations in Prolonged Starvation Minimize Protein Degradation
                                                                      • 30.3.2. Metabolic Derangements in Diabetes Result from Relative Insulin Insufficiency and Glucagon Excess
                                                                      • 30.3.3. Caloric Homeostasis: A Means of Regulating Body Weight
                                                                      • Metabolism Consists of Highly Interconnected Pathways
                                                                      • Each Organ Has a Unique Metabolic Profile
                                                                      • Food Intake and Starvation Induce Metabolic Changes
                                                                      • Fuel Choice During Exercise Is Determined by Intensity and Duration of Activity
                                                                      • Ethanol Alters Energy Metabolism in the Liver
                                                                      • Key Terms
                                                                      • Where to start
                                                                      • Books
                                                                      • Fuel metabolism
                                                                      • Metabolic adaptations in starvation
                                                                      • Diabetes mellitus
                                                                      • Exercise metabolism
                                                                      • Ethanol metabolism
                                                                      • 31.1. Prokaryotic DNA-Binding Proteins Bind Specifically to Regulatory Sites in Operons
                                                                        • 31.1.1. An Operon Consists of Regulatory Elements and Protein-Encoding Genes
                                                                        • 31.1.2. The lac Operator Has a Symmetric Base Sequence
                                                                        • 31.1.3. The lac Repressor Protein in the Absence of Lactose Binds to the Operator and Blocks Transcription
                                                                        • 31.1.4. Ligand Binding Can Induce Structural Changes in Regulatory Proteins
                                                                        • 31.1.5. The Operon Is a Common Regulatory Unit in Prokaryotes
                                                                        • 31.1.6. Transcription Can Be Stimulated by Proteins That Contact RNA Polymerase
                                                                        • 31.1.7. The Helix-Turn-Helix Motif Is Common to Many Prokaryotic DNA-Binding Proteins
                                                                        • 31.2.1. Nucleosomes Are Complexes of DNA and Histones
                                                                        • 31.2.2. Eukaryotic DNA Is Wrapped Around Histones to Form Nucleosomes
                                                                        • 31.2.3. The Control of Gene Expression Requires Chromatin Remodeling
                                                                        • 31.2.4. Enhancers Can Stimulate Transcription by Perturbing Chromatin Structure
                                                                        • 31.2.5. The Modification of DNA Can Alter Patterns of Gene Expression
                                                                        • 31.3.1. Steroids and Related Hydrophobic Molecules Pass Through Membranes and Bind to DNA-Binding Receptors
                                                                        • 31.3.2. Nuclear Hormone Receptors Regulate Transcription by Recruiting Coactivators and Corepressors to the Transcription Complex
                                                                        • 31.3.3. Steroid-Hormone Receptors Are Targets for Drugs
                                                                        • 31.3.4. Chromatin Structure Is Modulated Through Covalent Modifications of Histone Tails
                                                                        • 31.3.5. Histone Deacetylases Contribute to Transcriptional Repression
                                                                        • 31.3.6. Ligand Binding to Membrane Receptors Can Regulate Transcription Through Phosphorylation Cascades
                                                                        • 31.3.7. Chromatin Structure Effectively Decreases the Size of the Genome
                                                                        • 31.4.1. Attenuation Is a Prokaryotic Mechanism for Regulating Transcription Through Modulation of Nascent RNA Secondary Structure
                                                                        • 31.4.2. Genes Associated with Iron Metabolism Are Translationally Regulated in Animals
                                                                        • Prokaryotic DNA-Binding Proteins Bind Specifically to Regulatory Sites in Operons
                                                                        • The Greater Complexity of Eukaryotic Genomes Requires Elaborate Mechanisms for Gene Regulation
                                                                        • Transcriptional Activation and Repression Are Mediated by Protein-Protein Interactions
                                                                        • Gene Expression Can Be Controlled at Posttranscriptional Levels
                                                                        • Key Terms
                                                                        • Mechanism Problem
                                                                        • Chapter Integration Problem
                                                                        • Data Interpretation Problem
                                                                        • Where to start
                                                                        • Books
                                                                        • Prokaryotic gene regulation
                                                                        • Nucleosomes and histones
                                                                        • Nuclear hormone receptors
                                                                        • Chromatin and chromatin remodeling
                                                                        • Posttranscriptional regulation
                                                                        • Historical aspects
                                                                        • Chapter 32. Sensory Systems
                                                                          • 32.1. A Wide Variety of Organic Compounds Are Detected by Olfaction
                                                                            • 32.1.1. Olfaction Is Mediated by an Enormous Family of Seven-Transmembrane-Helix Receptors
                                                                            • 32.1.2. Odorants Are Decoded by a Combinatorial Mechanism
                                                                            • 32.1.3. Functional Magnetic Resonance Imaging Reveals Regions of the Brain Processing Sensory Information
                                                                            • 32.2.1. Sequencing the Human Genome Led to the Discovery of a Large Family of 7TM Bitter Receptors
                                                                            • 32.2.2. A Family of 7TM Receptors Almost Certainly Respond to Sweet Compounds
                                                                            • 32.2.3. Salty Tastes Are Detected Primarily by the Passage of Sodium Ions Through Channels
                                                                            • 32.2.4. Sour Tastes Arise from the Effects of Hydrogen Ions (Acids) on Channels
                                                                            • 32.2.5. Umami, the Taste of Glutamate, Is Detected by a Specialized Form of Glutamate Receptor
                                                                            • 32.3.1. Rhodopsin, a Specialized 7TM Receptor, Absorbs Visible Light
                                                                            • 32.3.2. Light Absorption Induces a Specific Isomerization of Bound 11-cis-Retinal
                                                                            • 32.3.3. Light-Induced Lowering of the Calcium Level Coordinates Recovery
                                                                            • 32.3.4. Color Vision Is Mediated by Three Cone Receptors That Are Homologs of Rhodopsin
                                                                            • 32.3.5. Rearrangements in the Genes for the Green and Red Pigments Lead to 𠇌olor Blindness”
                                                                            • 32.4.1. Hair Cells Use a Connected Bundle of Stereocilia to Detect Tiny Motions
                                                                            • 32.4.2. Mechanosensory Channels Have Been Identified in Drosophila and Bacteria
                                                                            • 32.5.1. Studies of Capsaicin, the Active Ingredient in “Hot” Peppers, Reveal a Receptor for Sensing High Temperatures and Other Painful Stimuli
                                                                            • 32.5.2. Subtle Sensory Systems Detect Other Environmental Factors Such as Earth's Magnetic Field
                                                                            • Smell, Taste, Vision, Hearing, and Touch Are Based on Signal-Transduction Pathways Activated by Signals from the Environment
                                                                            • A Wide Variety of Organic Compounds Are Detected by Olfaction
                                                                            • Taste Is a Combination of Senses That Function by Different Mechanisms
                                                                            • Photoreceptor Molecules in the Eye Detect Visible Light
                                                                            • Hearing Depends on the Speedy Detection of Mechanical Stimuli
                                                                            • Touch Includes the Sensing of Pressure, Temperature, and Other Factors
                                                                            • Key Terms
                                                                            • Chapter Integration Problem
                                                                            • Mechanism Problem
                                                                            • Media Problems
                                                                            • Where to start
                                                                            • Olfaction
                                                                            • Taste
                                                                            • Vision
                                                                            • Hearing
                                                                            • Touch and pain reception
                                                                            • Other sensory systems
                                                                            • 33.1. Antibodies Possess Distinct Antigen-Binding and Effector Units
                                                                            • 33.2. The Immunoglobulin Fold Consists of a Beta-Sandwich Framework with Hypervariable Loops
                                                                            • 33.3. Antibodies Bind Specific Molecules Through Their Hypervariable Loops
                                                                              • 33.3.1. X-Ray Analyses Have Revealed How Antibodies Bind Antigens
                                                                              • 33.3.2. Large Antigens Bind Antibodies with Numerous Interactions
                                                                              • 33.4.1. J (Joining) Genes and D (Diversity) Genes Increase Antibody Diversity
                                                                              • 33.4.2. More Than 10 8 Antibodies Can Be Formed by Combinatorial Association and Somatic Mutation
                                                                              • 33.4.3. The Oligomerization of Antibodies Expressed on the Surface of Immature B Cells Triggers Antibody Secretion
                                                                              • 33.4.4. Different Classes of Antibodies Are Formed by the Hopping of VH Genes
                                                                              • 33.5.1. Peptides Presented by MHC Proteins Occupy a Deep Groove Flanked by Alpha Helices
                                                                              • 33.5.2. T-Cell Receptors Are Antibody-like Proteins Containing Variable and Constant Regions
                                                                              • 33.5.3. CD8 on Cytotoxic T Cells Acts in Concert with T-Cell Receptors
                                                                              • 33.5.4. Helper T Cells Stimulate Cells That Display Foreign Peptides Bound to Class II MHC Proteins
                                                                              • 33.5.5. Helper T Cells Rely on the T-Cell Receptor and CD4 to Recognize Foreign Peptides on Antigen-Presenting Cells
                                                                              • 33.5.6. MHC Proteins Are Highly Diverse
                                                                              • 33.5.7. Human Immunodeficiency Viruses Subvert the Immune System by Destroying Helper T Cells
                                                                              • 33.6.1. T Cells Are Subject to Positive and Negative Selection in the Thymus
                                                                              • 33.6.2. Autoimmune Diseases Result from the Generation of Immune Responses Against Self-Antigens
                                                                              • 33.6.3. The Immune System Plays a Role in Cancer Prevention
                                                                              • Antibodies Possess Distinct Antigen-Binding and Effector Units
                                                                              • The Immunoglobulin Fold Consists of a Beta-Sandwich Framework with Hypervariable Loops
                                                                              • Antibodies Bind Specific Molecules Through Their Hypervariable Loops
                                                                              • Diversity Is Generated by Gene Rearrangements
                                                                              • Major-Histocompatibility-Complex Proteins Present Peptide Antigens on Cell Surfaces for Recognition by T-Cell Receptors
                                                                              • Immune Responses Against Self-Antigens Are Suppressed
                                                                              • Key Terms
                                                                              • Mechanism Problem
                                                                              • Chapter Integration Problem
                                                                              • Data Interpretation Problem
                                                                              • Where to start
                                                                              • Books
                                                                              • Structure of antibodies and antibody-antigen complexes
                                                                              • Generation of diversity
                                                                              • MHC proteins and antigen processing
                                                                              • T-cell receptors and signaling complexes
                                                                              • HIV and AIDS
                                                                              • Discovery of major concepts
                                                                              • 34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
                                                                                • 34.1.1. A Motor Protein Consists of an ATPase Core and an Extended Structure
                                                                                • 34.1.2. ATP Binding and Hydrolysis Induce Changes in the Conformation and Binding Affinity of Motor Proteins
                                                                                • 34.2.1. Muscle Is a Complex of Myosin and Actin
                                                                                • 34.2.2. Actin Is a Polar, Self-Assembling, Dynamic Polymer
                                                                                • 34.2.3. Motions of Single Motor Proteins Can Be Directly Observed
                                                                                • 34.2.4. Phosphate Release Triggers the Myosin Power Stroke
                                                                                • 34.2.5. The Length of the Lever Arm Determines Motor Velocity
                                                                                • 34.3.1. Microtubules Are Hollow Cylindrical Polymers
                                                                                • 34.3.2. Kinesin Motion Is Highly Processive
                                                                                • 34.3.3. Small Structural Changes Can Reverse Motor Polarity
                                                                                • 34.4.1. Bacteria Swim by Rotating Their Flagella
                                                                                • 34.4.2. Proton Flow Drives Bacterial Flagellar Rotation
                                                                                • 34.4.3. Bacterial Chemotaxis Depends on Reversal of the Direction of Flagellar Rotation
                                                                                • Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
                                                                                • Myosins Move Along Actin Filaments
                                                                                • Kinesin and Dynein Move Along Microtubules
                                                                                • A Rotary Motor Drives Bacterial Motion
                                                                                • Key Terms
                                                                                • Mechanism Problem
                                                                                • Chapter Integration Problem
                                                                                • Data Interpretation Problem
                                                                                • Where to start
                                                                                • Books
                                                                                • Myosin and actin
                                                                                • Kinesin, dynein, and microtubules
                                                                                • Bacterial motion and chemotaxis
                                                                                • Historical aspects
                                                                                • Appendix A: Physical Constants and Conversion of Units
                                                                                • Appendix B: Acidity Constants
                                                                                • Appendix C: Standard Bond Lengths

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                                                                                Reverse oxidative phosphorylation? - Biology

                                                                                Reactivity in Chemistry

                                                                                PS1. Introduction to Photosynthesis

                                                                                In biology, energy is needed in order to drive all sorts of biochemical processes. Energy is needed to stay alive. There are plenty of energy sources on earth. Tectonic forces release massive amounts of heat and drive the conversion of some minerals into gaseous products for example, metal sulfides such as zincblende can be converted to gaseous hydrogen sulfide, H2S. Sometimes, all that heat and gas finds its way to the earth's surface in the form of volcanoes. In the oceans, some marine organisms get their energy from gases released from volcanic vents, such as methane and hydrogen sulfide. However, sunlight is an even more abundant source of energy over most of the earth's surface. In photosynthesis, light energy is absorbed and used to make ATP. Remember, ATP is like a portable battery pack in biology it can travel to different parts of a cell where it can be used to power uphill steps in biochemical reactions.

                                                                                Figure PS1.1. Absorption of photons from the sun is coupled to production of ATP, the biological battery pack.

                                                                                Plants, algae, and some bacteria are capable of carrying out photosynthesis. They could get the immediate benefit of portable ATP molecules to drive biochemical reactions. However, the production of ATP in photosynthesis is also connected to carbon capture. Carbon dioxide from the air is incorporated into carbohydrate molecules. This conversion happens in a series of reactions called "dark reactions", because they keep happening even without sunlight. The carbohydrates can be stored, long-term, and later they can be used as energy sources via glycolysis and the citric acid cycle.

                                                                                Figure PS1.1. In photosynthesis, the ATP is diverted to carbohydrate production for long-term energy storage.

                                                                                Animals, of course, benefit from this process indirectly because they can also use carbohydrates as a source of energy. The breakdown of carbohydrates releases energy through the usual trade-off: slightly weaker C-H and C-C bonds are broken and slightly stronger O-H and C-O bonds are made, meaning there is an overall release of energy. By eating plants, we can immediately access these carbohydrates without all the fuss of standing in the sun all day making them ourselves.

                                                                                If you remember some basic plant biology, you may be familar with another aspect of photosynthesis. The "balanced reaction" for photosynthesis also involves the conversion of water to molecular oxygen, as follows:

                                                                                Oxygen is a key player in oxidative phosphorylation, in which glycolysis and the TCA cycle are made even more efficient by boosting the amount of ATP produced for every glucose molecule broken down. Most organisms (including us) just can't survive without that extra ATP we depend on plants for our survival in more ways than one.

                                                                                But in contrast to what is suggested by the balanced reaction, the production of oxygen by plants is actually carried out separately from carbohydrate synthesis. Production of oxygen is actually part of the "light reaction", along with ATP synthesis.

                                                                                Figure PS1.3. Photosynthesis is also associated with the production of molecular oxygen.

                                                                                In photosynthesis, water is oxidised to produce molecular oxygen. Plants take the electrons that they have stripped from the water molecules and divert them into an electron transport chain. Energy harnessed by that electron transport chain is used to convert ADP to ATP. In oxidative phosphorylation, organisms (including plants) take electrons from NADH and succinate and divert them into an electron transport chain, eventually depositing them onto an oxygen molecule to make water. Energy harnessed by that electron transport chain is used to convert ADP to ATP.

                                                                                That means we have two opposite processes that both are harnessed to produce ATP. In one process, the electrons run downhill energetically and are deposited on dioxygen to make water. That's oxidative phosphorylation. Photosynthesis is really oxidative phosphorylation running in reverse: the electrons start on water and proceed through an electron chain from there. But if oxidative phosphorylation runs downhill, then photosynthesis must run uphill.

                                                                                That's where the light comes in. The light absorbed in photosynthesis is used to lift the electrons uphill in energy from there, they can start rolling downhill through the electron transport chain, releasing energy along the way that can be harnessed for ATP formation.

                                                                                All of these events take place in a special organelle in the plant called the chloroplast. Chloroplasts are a little bit like mitochondria, where the important metabolic processes such the TCA cycle and oxidative phosphorylation take place. Like mitochondria, chloroplasts contain their own DNA and ribosomes for protein production and they are passed on directly from mother cell to daughter cell. Chloroplasts have a double membrane and are filled with an aqueous medium called the stroma.

                                                                                Figure PS1.4. Simplified diagram of a chloroplast.

                                                                                Within the chloroplast there are structures called thylakoids. A thylakoid is like a complex water balloon it has a membrane and is filled with an aqueous medium called the lumen. Unlike a simple water balloon, though, the thylakoid has portions that are deeply folded, so that they look like discs stacked in layers. These portions of the thylakoid are called the grana. The regular, non-folded portions are called the lamellae.

                                                                                Figure PS1.5. Simplified diagram of thylakoid structure.

                                                                                The thylakoid plays a very important role in phtosynthesis. A group of protein complexes bound to the thylakoid membrane carry out the absorption of light energy, the conversion of water to dioxygen, and the production of ATP, as well as an electron carrier, NADPH. The ATP and NADPH are released into the surrounding stroma. A soluble protein in the stroma, called ribulose bisphosphate carboxylase (RuBisCo) captures carbon dioxide and covalently attaches it to a carbohydrate molecule. Other proteins then use the ATP and NADPH to reduce the carboxylate group (from the CO2) into a regular part of the carbohydrate chain. In this way, rather than trying to knit six carbon dioxide molecules together into a glucose, the problem is simplified into just taking up one carbon dioxide at a time, adding it into a pre-existing sugar.

                                                                                The ATP is produced by an ATP synthase, which is very similar to the complex used for the same purpose during oxidative phosphorylation. Just like the ATP synthase in the mitochondria, this one is driven by a proton gradient. The proton gradient is created through an electron transport pathway, just like the one in the mitochondria. In fact, many of the features of photosynthesis are pretty similar to oxidative phosphorylation. A crucial difference is that the electron transport chain in oxidative phosphorylation starts with NADH and ends with water, whereas in photosynthesis it is the reverse: the chain starts with water and ends with NADPH. The electron transport chain in oxidative phosphorylation is exothermic, running downhill in energy. The electron transport chain in photosynthesis would be endothermic, but can be sustained by the input of energy in the form of light.

                                                                                Figure PS1.6. Simplified diagram of the major participants in photosynthesis.

                                                                                See the section on photosynthesis at Henry Jakubowski's Biochemistry Online.

                                                                                This site is written and maintained by Chris P. Schaller, Ph.D., College of Saint Benedict / Saint John's University (with contributions from other authors as noted). It is freely available for educational use.

                                                                                Structure & Reactivity in Organic, Biological and Inorganic Chemistry by Chris Schaller is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License.

                                                                                Send corrections to [email protected]

                                                                                This material is based upon work supported by the National Science Foundation under Grant No. 1043566.

                                                                                Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

                                                                                What are the similarities and differences between photophosphorylation and oxidative phosphorylation?

                                                                                Both processes operate on the same basic principle passing electrons down a chain in order to create a proton (H + ) gradient, allowing the formation of ATP. The biggest difference is where they occur. Photophosphorylation occurs in the thylakoid membrane of chloroplasts during the light-dependent stage of photosynthesis. Light in the form of photons supplies the energy needed to excite two e - s in PSII (photosystem II), which are then passed along the transport chain. Oxidative phosphorylationoccurs in the membrane of mitochondrial christae during cellular respiration. Here, the e - s are supplied by NAD and FAD, with oxygen acting as the last electron acceptor, leading to the formation of H2O. During photophosphorylation, NADP acts as the last e - acceptor , leading to the formation of NADPH.

                                                                                In both processes e - s are passed down a chain of electron transfer agents in a series of redox reactions. In both reactions, as e - s are passed along the cytochrome complex, H + ions are pumped from an area of low to high concentration, creating a proton gradient. During photophosphorylation e - s are pumped from the stroma into the thylakoid, while in oxidative phosphorylation e - s are pumped from the matrix into the intermembranal space. The chemiosmosis of H+ ions down the concentration gradient through the pores of ATPsynthase then supplies the energy needed to phosphorylate ADP into ATP, which is the primary "energy carrier" in cells.

                                                                                Watch the video: 25. Oxidative Phosphorylation (August 2022).