Biochemistry, 4th Edition P88 pps

26.8 How Are Thymine Nucleotides Synthesized? 833 enzyme must be turned on and off in response to the need for dNTPs. Second, the relative amounts of each NDP substrate transformed into dNDP must be con- trolled so that the right balance of dATPϺdGTPϺdCTPϺdTTP is produced. The two different effector-binding sites on ribonucleotide reductase, discrete from the substrate-binding catalytic site, are designed to serve these purposes. As noted previ- ously, these two regulatory sites are designated the overall activity site and the substrate specificity site. Only ATP and dATP are able to bind at the overall activity site. ATP is an allosteric activator and dATP is an allosteric inhibitor, and they compete for the same site. If ATP is bound, the enzyme is active, whereas if its deoxy counterpart, dATP, occupies this site, the enzyme is inactive. That is, ATP is a positive effector and dATP is a negative effector with respect to enzyme activity, and they compete for the same site. The second regulatory site, the substrate specificity site, can bind either ATP, dTTP, dGTP, or dATP, and the substrate specificity of the enzyme is determined by which of these nucleotides occupies this site. If ATP is in the substrate specificity site, ribonucleotide reductase preferentially binds pyrimidine nucleotides (UDP or CDP) at its active site and reduces them to dUDP and dCDP. With dTTP in the specificity-determining site, GDP is the preferred substrate. When dGTP binds to the specificity site, ADP becomes the favored substrate for reduction. The rationale for these varying affinities is as follows (Figure 26.23): High [ATP] is consistent with cell growth and division and, consequently, the need for DNA synthesis. Thus, ATP binds in the overall activity site of ribonucleotide reductase, turning it on and promoting production of dNTPs for DNA synthesis. Under these conditions, ATP is also likely to occupy the substrate specificity site, so UDP and CDP are bound at the catalytic site and reduced to dUDP and dCDP. Both of these pyrimidine deoxynucleoside diphosphates are precursors to dTTP. Thus, elevation of dUDP and dCDP levels leads to an increase in [dTTP]. High dTTP levels increase the likelihood that it will occupy the substrate specificity site, in which case GDP becomes the preferred substrate and dGTP levels rise. Upon dGTP association with the substrate specificity site, ADP is the favored substrate, leading to ADP reduction and the eventual accumulation of dATP. Binding of dATP to the overall activity site then shuts the enzyme down. In summary, the relative affinities of the three classes of nucleotide binding sites in ribonucleotide reductase for the various substrates, activators, and inhibitors are such that the formation of dNDPs proceeds in an orderly and balanced fashion. As these dNDPs are formed in amounts consistent with cellular needs, their phosphorylation by nucleoside diphosphate kinases produces dNTPs, the actual substrates of DNA synthesis. 26.8 How Are Thymine Nucleotides Synthesized? The synthesis of thymine nucleotides proceeds from other pyrimidine deoxyribonucleotides. Cells have no requirement for free thymine ribonucleotides and do not synthesize them. Small amounts of thymine ribonucleotides do occur in tRNA (tRNA is notable for having unusual nucleotides), but these Ts arise via methylation of U residues already incorporated into the tRNA. Both dUDP and 1 2 3 4 5 6 Energy status of cell is robust; [ATP] is high. Make DNA: ATP occupies activity site A: ribonucleotide reductase ON ATP in specificity site S favors CDP or UDP in catalytic site C [dCDP], [dUDP] dCDP dUDP dUMP dTMP dTTP dTTP occupies specificity site S, favoring GDP or ADP in catalytic site C dGTP occupies specificity site S, favoring ADP in catalytic site C [dADP] dATP replaces ATP in activity site A: ribonucleotide reductase OFF GDP dGDP dGTP FIGURE 26.23 Regulation of deoxynucleotide biosynthesis: the rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribonucleotide reductase. 834 Chapter 26 Synthesis and Degradation of Nucleotides dCDP can lead to formation of dUMP, the immediate precursor for dTMP synthesis (Figure 26.24). Interestingly, formation of dUMP from dUDP passes through dUTP, which is then cleaved by dUTPase, a pyrophosphatase that re- moves PP i from dUTP. The action of dUTPase prevents dUTP from serving as a substrate in DNA synthesis. An alternative route to dUMP formation starts with From dUDP: dUDP From dCDP: dCDP dUTP dCMP dUMP dUMP dTMP dTMP FIGURE 26.24 Pathways of dTMP synthesis. dTMP production is dependent on dUMP formation from dCDP and dUDP. A DEEPER LOOK Fluoro-Substituted Analogs as Therapeutic Agents Carbon–fluorine bonds are exceedingly rare in nature, and fluorine is an uncommon constituent of biological molecules. F has three properties attractive to drug designers: (1) It is the smallest replacement for an H atom in organic synthesis, (2) fluorine is the most electronegative element, and (3) the FOC bond is rela- tively unreactive. This steric compactness and potential for strong inductive effects through its electronegativity renders F a useful substituent in the construction of inhibitory analogs of enzyme substrates. One interesting strategy is to devise fluorinated precursors that are taken up and processed by normal metabolic pathways to generate a potent antimetabolite. A classic example is fluoroacetate. FCH 2 COO Ϫ is exceptionally toxic because it is readily converted to fluorocitrate by citrate synthase of the citric acid cycle (see Chapter 19). In turn, fluorocitrate is a powerful inhibitor of aconitase. The metabolic transformation of an other- wise innocuous compound into a poisonous derivative is termed lethal synthesis. 5-Fluorouracil and 5-fluorocytosine are also examples of this strategy (see Human Biochemistry on page 835). Unlike hydrogen, which is often abstracted from substrates as H ϩ , electronegative fluorine cannot be readily eliminated as the corresponding F ϩ . Thus, enzyme inhibitors can be fashioned in which F replaces H at positions where catalysis involves H removal as H ϩ . Thymidylate synthase catalyzes removal of H from dUMP as H ϩ through a covalent catalysis mechanism. A thiol group on this enzyme normally attacks the 6-position of the uracil moiety of 2Ј-deoxyuridylic acid so that C-5 can act as a carbanion in attack on the methylene carbon of N 5 ,N 10 -methylene-THF (see accompa- nying figure). Regeneration of free enzyme then occurs through loss of the C-5 H atom as H ϩ and dissociation of product dTMP. If F replaces H at C-5 as in 2Ј-deoxy-5-fluorouridylate (FdUMP), the enzyme is immobilized in a very stable ternary [enzymeϺFdUMPϺ methylene-THF] complex and effectively inactivated. Enzyme inhibitors like FdUMP whose adverse properties are elicited only through direct participation in the catalytic cycle are variously called mechanism-based inhibitors, suicide substrates, or Trojan horse substrates. + dR dR dR H H 2 N N O N N N H H CH 2 NH 2 C R H 2 3 4 1 8 7 6 9 10 5 N N F H 2 3 4 1 6 5 O H O Cys SH N N F H 2 3 4 1 6 5 O H O – S Cys N N H O H O S Cys CH 2 F N H 2 N N O N N N H H H CH 2 H 2 3 4 1 8 7 6 9 10 5 E E E R H H H N 5 ,N 10 -methylene- THF Ternary com p lex ᮣ The effect of the 5-fluoro substitution on the mechanism of action of thymidylate synthase. An enzyme thiol group (from a Cys side chain) ordinarily attacks the 6-position of dUMP so that C-5 can react as a carbanion with N 5 ,N 10 -methylene-THF. Normally, free enzyme is regenerated following release of the hydrogen at C-5 as a proton. Because release of fluorine as F ϩ cannot occur, the ternary (three-part) complex of [enzymeϺfluorouridylateϺmethylene-THF] is stable and per- sists, preventing enzyme turnover. (The N 5 ,N 10 -methylene-THF structure is given in abbreviated form.) 26.8 How Are Thymine Nucleotides Synthesized? 835 dCDP, which is dephosphorylated to dCMP and then deaminated by dCMP deaminase (Figure 26.25), leaving dUMP. dCMP deaminase provides a second point for allosteric regulation of dNTP synthesis; it is allosterically activated by dCTP and feedback-inhibited by dTTP. Of the four dNTPs, only dCTP does not interact with either of the regulatory sites on ribonucleotide reductase (see Fig- ure 26.20). Instead, it acts upon dCMP deaminase. Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase (Figure 26.26). This enzyme methylates dUMP at the 5-position to create dTMP; the methyl donor is the one-carbon folic acid derivative N 5 ,N 10 -methylene-THF. The reaction is actually a reductive methylation in which the one-carbon unit is trans- ferred at the methylene level of reduction and then reduced to the methyl level. The THF cofactor is oxidized at the expense of methylene reduction to yield DHF. DHFR then reduces DHF back to THF for service again as a one-carbon vehicle (see panel a of the figure in A Deeper Look on page 816). Thymidylate synthase sits at a junction connecting dNTP synthesis with folate metabolism. dCMP deaminase P CH 2 P CH 2 NH 2 O O N N HH HOH dCMP NH 4 + dCTP dTTP O O N N HH HOH dUMP O H + H + + H 2 O (a) (b) FIGURE 26.25 (a) The dCMP deaminase reaction. (b) Trimeric dCMP deaminase. Each chain has a bound dCTP molecule (purple) and a Mg 2ϩ ion (orange) (pdb id ϭ 1XS4). HUMAN BIOCHEMISTRY Fluoro-Substituted Pyrimidines in Cancer Chemotherapy, Fungal Infections, and Malaria 5-Fluorouracil (5-FU; see part a of the figure) is a thymine analog. It is converted in vivo to 5Ј-fluorouridylate by a PRPP-dependent phosphoribosyltransferase and passes through the reactions of dNTP synthesis, culminating ultimately as 2Ј-deoxy-5-fluorouridylic acid, a potent inhibitor of dTMP synthase (see A Deeper Look on page 834). 5-FU is used as a chemotherapeutic agent in the treatment of human cancers. Similarly, 5-fluorocytosine (see part b) is used as an antifungal drug because fungi, unlike mammals, can convert it to 2Ј-deoxy-5-fluorouridylate. Furthermore, malarial parasites can use exogenous orotate to make pyrimidines for nucleic acid synthesis, whereas mammals cannot. Thus, 5-fluoroorotate (see part c) is an effective antimalarial drug because it is selectively toxic to these parasites. NH 2 O – O H N N O F H O N N F H O H N N F H O CO 5-Fluorouracil 5-Fluorocytosine 5-Fluoroorotate (a) (c) (b) SUMMARY 26.1 Can Cells Synthesize Nucleotides? Nucleotides are ubiquitous constituents of life and nearly all cells are capable of synthesizing them “from scratch” via de novo pathways. Rapidly proliferating cells must make lots of purine and pyrimidine nucleotides to satisfy demands for DNA and RNA synthesis. Nucleotide biosynthetic pathways are attractive targets for the clinical control of rapidly dividing cells such as cancers or infectious bacteria. Many antibiotics and anticancer drugs are inhibitors of purine or pyrimidine nucleotide biosynthesis. 26.2 How Do Cells Synthesize Purines? The nine atoms of the purine ring system are derived from aspartate (N-1), glutamine (N-3 and N-9), glycine (C-4, C-5, and N-7), CO 2 (C-6), and THF one-carbon derivatives (C-2 and C-8). The atoms of the purine ring are successively added to ribose-5-phosphate, so purines begin as nucleotide derivatives through as- sembly of the purine ring system directly on the ribose. Because purine biosynthesis depends on folic acid derivatives, it is sensitive to inhibition by folate analogs. Distinct, two-step metabolic pathways diverge from IMP, one leading to AMP and the other to GMP. Purine biosynthesis is regulated at several stages: Reaction 1 (ribose-5-phosphate pyrophosphoki- nase) is feedback-inhibited by ADP and GDP; the enzyme catalyzing reaction 2 (glutamine phosphoribosyl pyrophosphate amidotransferase) has two inhibitory allosteric sites, one where adenine nucleotides bind and another where guanine nucleotides bind. PRPP is a “feed-forward” activator of this enzyme. The first reaction in the conversion of IMP to AMP involves adenylosuccinate synthetase, which is inhibited by AMP; the first step in the conversion of IMP to GMP is catalyzed by IMP dehydro- genase and is inhibited by GMP. ATP-dependent kinases form nucleoside diphosphates and triphosphates from AMP and GMP. 26.3 Can Cells Salvage Purines? Purine ring systems represent a metabolic investment by cells, and salvage pathways exist to recover them when degradation of nucleic acids releases free purines in the form of adenine, guanine, and hypoxanthine (the base in IMP). Hypoxanthine- guanine phosphoribosyltransferase (HGPRT) acts on either hypoxan- 836 Chapter 26 Synthesis and Degradation of Nucleotides It has become a preferred target for inhibitors designed to disrupt DNA synthesis. An indirect approach is to employ folic acid precursors or analogs as anti- metabolites of dTMP synthesis (see panel b of the figure in A Deeper Look on page 818). Purine synthesis is affected as well because it is also dependent on THF (see Figure 26.3). dR dUMP dTMP O O – H N N O H H 2 N N O N N N H H CH 2 NH 2 C R H H 2 3 4 1 8 7 6 9 10 5 H H 2 N N O N N N H H CH 2 N H R H H H H R O H N N O CH 3 H H 2 N N O N N N H H CH 2 N R H O CCH 2 + H 3 N Serine O – O CCH + H 3 N CH 2 OH N 5 ,N 10 -methylene-THF Thymidylate synthase Dihydrofolic acid (DHF) Serine hydroxymethyl- transferase Dihydrofolate reductase Tetrahydrofolic acid (THF) (a) NADP + Glycine + H 2 O NADPH + H + (b) FIGURE 26.26 (a) The thymidylate synthase reaction. The 5-CH 3 group is ultimately derived from the ␤-carbon of serine. (b) Thymidylate synthase dimer. Each monomer has a bound folate analog (green) and dUMP (light blue) (pdb id ϭ 1JUJ). Problems 837 PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. Draw the purine and pyrimidine ring structures, indicating the metabolic source of each atom in the rings. 2. Starting from glutamine, aspartate, glycine, CO 2 and N 10 -formyl- THF, how many ATP equivalents are expended in the synthesis of (a) ATP, (b) GTP, (c) UTP, and (d) CTP? 3. Illustrate the key points of regulation in (a) the biosynthesis of IMP, AMP, and GMP; (b) E. coli pyrimidine biosynthesis; and (c) mam- malian pyrimidine biosynthesis. 4. Indicate which reactions of purine or pyrimidine metabolism are affected by the inhibitors (a) azaserine, (b) methotrexate, (c) sulfon- amides, (d) allopurinol, and (e) 5-fluorouracil. 5. Since dUTP is not a normal component of DNA, why do you sup- pose ribonucleotide reductase has the capacity to convert UDP to dUDP? 6. Describe the underlying rationale for the regulatory effects exerted on ribonucleotide reductase by ATP, dATP, dTTP, and dGTP. 7. (Integrates with Chapters 18–20 and 22.) By what pathway(s) does the ribose released upon nucleotide degradation enter intermediary metabolism and become converted to cellular energy? How many ATP equivalents can be recovered from one equivalent of ribose? 8. (Integrates with Chapter 25.) At which steps does the purine biosynthetic pathway resemble the pathway for biosynthesis of the amino acid histidine? 9. Write reasonable chemical mechanisms for steps 6, 8, and 9 in purine biosynthesis (see Figure 26.3). 10. Write a balanced equation for the conversion of aspartate to fu- marate by the purine nucleoside cycle in skeletal muscle. 11. Write a balanced equation for the oxidation of uric acid to glyoxylic acid, CO 2 , and NH 3 , showing each step in the process and naming all of the enzymes involved. 12. (Integrates with Chapter 15.) E. coli aspartate transcarbamoylase (ATCase) displays classic allosteric behavior. This ␣ 6 ␤ 6 enzyme is activated by ATP and feedback-inhibited by CTP. In analogy with the behavior of glycogen phosphorylase shown in Figure 15.14, illustrate the allosteric v versus [aspartate] curves for ATCase (a) in the absence of effectors, (b) in the presence of CTP, and (c) in the presence of ATP. *13. (Integrates with Chapter 15.) Unlike its allosteric counterpart glycogen phosphorylase (an ␣ 2 enzyme), E. coli ATCase has a heteromeric (␣ 6 ␤ 6 ) organization. The ␣-subunits bind aspartate and are considered catalytic subunits, whereas the ␤-subunits bind CTP or ATP and are considered regulatory subunits. How would you describe the subunit organization of ATCase from a functional point of view? 14. (Integrates with Chapter 20.) Starting from HCO 3 Ϫ , glutamine, aspartate, and ribose-5-P, how many ATP equivalents are consumed in the synthesis of dTTP in a eukaryotic cell, assuming dihydroorotate oxidation is coupled to oxidative phosphorylation? How does this result compare with the ATP costs of purine nucleotide biosynthesis calculated in problem 2? 15. Write a balanced equation for the synthesis of dTMP from UMP and N 5 ,N 10 -methylene-THF. Thymidylate synthase has four active-site arginine residues (Arg 23 , Arg 178Ј , Arg 179Ј , and Arg 218 ) involved in substrate binding. Postulate a role for the side chains of these Arg residues. 16. Enzymes that bind phosphoribosyl-5-phosphate (PRPP) have a com- mon structural fold, the PRT fold, which unites them as a structural family. PRT here refers to the phosphoribosyl transferase activity displayed by some family members. Typically, in such reactions, PP i is displaced from PRPP by a nitrogen-containing nucleophile. Several such reactions occur in purine metabolism. Identify two such reactions where the enzyme involved is likely to be a PRT family member. 17. The crystal structure of E. coli dihydrofolate reductase (DFR) with NADP ϩ and folate bound can be found in the Protein Data Bank thine to form IMP or guanine to form GMP; an absence of HGPRT is the basis of Lesch-Nyhan syndrome. 26.4 How Are Purines Degraded? Dietary nucleic acids are digested to nucleotides by various nucleases and phosphodiesterases, the nucleotides are converted to nucleosides by base-specific nucleotidases and nonspecific phosphatases, and then nucleosides are hydrolyzed to release the purine base. Only the pentoses of nucleotides serve as sources of metabolic energy. In humans, the purine ring is oxidized to uric acid by xanthine oxidase and excreted. Gout occurs when bodily fluids accumulate an excess of uric acid. Skeletal muscle operates a purine nucleoside cycle as an anaplerotic pathway. 26.5 How Do Cells Synthesize Pyrimidines? In contrast to formation of the purine ring system, the pyrimidine ring system is completed before a ribose-5-P moiety is attached. Only two precursors, carbamoyl-P and aspartate, contribute atoms to the six-membered pyrimidine ring. The first step in humans is catalyzed by CPS-II. ATCase then links carbamoyl-P with aspartate. Subsequent reactions close the ring and oxidize it before adding ribose-5-P, using ␣-PRPP as donor. Decarboxylation gives UMP. In mammals, the six enzymatic activities of pyrimidine biosynthesis are distributed among only three proteins, two of which are multifunctional polypeptides. Purine and pyrimidine synthesis in mammals are two prominent examples of metabolic channeling. UMP leads to UTP, the substrate for formation of CTP via CTP synthetase. Regulation of pyrimidine synthesis in animals occurs at CPS-II. UDP and UTP are feedback inhibitors, whereas PRPP and ATP are allosteric activators. In bacteria, regulation acts at ATCase through feedback inhibition by CTP (or UTP) and activation by ATP. 26.6 How Are Pyrimidines Degraded? Degradation of the pyrimidine ring generates ␤-alanine, CO 2 , and ammonia. In humans, pyrimidines are recycled from nucleosides, but free pyrimidine bases are not salvaged. 26.7 How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? 2Ј-Deoxyribonucleotides are formed from ribonucleotides through reduction at the 2Ј-position of the ribose ring in NDPs. The reaction, catalyzed by ribonucleotide reductase, involves a free radical mechanism that replaces the 2Ј-OH by a hydride ion (HϺ Ϫ ). Thiore- doxin provides the reducing power for ribonucleotide reduction. Class Ia ribonucleotide reductases have three different nucleotide-binding sites: the catalytic site (or active site), which binds substrates (ADP, CDP, GDP, and UDP); the substrate specificity site, which can bind ATP, dATP, dGTP, or dTTP; and the overall activity site, which binds either the activator ATP or the negative effector dATP. The relative affinities of the three classes of nucleotide binding sites in ribonucleotide reductase for the various substrates, activators, and inhibitors are such that the various dNDPs are formed in amounts consistent with cellular needs. 26.8 How Are Thymine Nucleotides Synthesized? Both dUDP and dCDP can lead to formation of dUMP, the immediate precursor for dTMP synthesis. Formation of dTMP from dUMP is catalyzed by thymidylate synthase through reductive methylation of dUMP at the 5-position. The methyl donor is the one-carbon folic acid derivative N 5 ,N 10 -methylene- THF. Fluoro-substituted pyrimidine analogs such as 5-fluorouracil (5-FU), 5-fluorocytosine, and 5-fluoroorotate can be converted to FdUMP, which inhibits thymidylate synthase. These fluoro compounds have found a range of therapeutic uses in treating diseases from cancer to malaria. 838 Chapter 26 Synthesis and Degradation of Nucleotides (www.rcsb.org/pdb) as file 7DFR. Go to this website, enter “7DFR” in the search line, and click on “KiNG” under “Display options” when the 7DFR page comes up. Explore the KiNG graphic of the DFR structure to visualize how its substrates are bound. (If you hold down the left button on your mouse and move the cursor over the image, you can rotate the structure to view it from different per- spectives.) Note in particular the spatial relationship between the nicotinamide ring of NADP ϩ and the pterin ring of folate. Do you now have a better appreciation for how this enzyme works? Note also the location of polar groups on the two substrates in relation to the DFR structure. 18. E. coli aspartate transcarbamoylase is an allosteric enzyme (see problem 12) composed of six catalytic (C) subunits and six regulatory (R) subunits. Protein Data Bank file 1RAA shows one-third of the ATCase holoenzyme (two C subunits and two R subunits; CTP molecules are bound to the R subunits). Explore this structure using the KiNG display option. What secondary structural motif dom- inates the R subunit structure? Protein Data Bank file 2IPO also shows one-third of the ATCase holoenzyme (two C subunits and two R subunits), but in this structure molecules of the substrate analog N-(2-phosphonoacetyl)-L-asparagine are bound to the C subunits. Explore this structure using the KiNG display option. Note the distance separating the ATCase active site from its allosteric site. Interpret what you see in terms of the Monod– Wyman–Changeux model for allosteric regulation (see Chapter 15). Which of these structures corresponds to the MWC R-state, and which corresponds to the T-state? Preparing for the MCAT Exam 19. Examine Figure 26.6 and predict the relative rates of the regulated reactions in the purine biosynthetic pathway from ribose-5-P to GMP and AMP under conditions in which GMP levels are very high. 20. Decide from Figures 18.1, 25.31, 26.26, and the Deeper Look box on page 817 which carbon atom(s) in glucose would be most likely to end up as the 5-CH 3 carbon in dTMP. FURTHER READING Purine Metabolism Kisker, C., Schindelin, H., and Rees, D. C., 1997. Molybdenum-containing enzymes: Structure and mechanism. Annual Review of Biochemistry 66:233–267. Mueller, E. J., et al., 1994. N 5 -carboxyaminoimidazole ribonucleotide: Evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli. Biochem- istry 33:2269–2278. Watts, R. W. E., 1983. Some regulatory and integrative aspects of purine nucleotide synthesis and its control: An overview. Advances in Enzyme Regulation 21:33–51. Wilson, D. K., Rudolph, F. B., and Quiocho, F. A., 1991. Atomic structure of adenosine deaminase complexed with a transition-state analog: Understanding catalysis and immunodeficient mutations. Science 252:1279–1284. Pyrimidine Metabolism Connolly, G. P., and Duley, J. A., 1999. Uridine and its nucleotides: Bio- logical actions, therapeutical potentials. Trends in Phrmacological Sci- ences 20:218–225. Graves, L. M., et al., 2000. Regulation of carbamoyl phosphate synthetase by MAP kinase. Nature 403:328–331. Jones, M. E., 1980. Pyrimidine nucleotide biosynthesis in animals: Genes, enzymes and regulation of UMP biosynthesis. Annual Review of Bio- chemistry 49:253–279. Metabolic Disorders of Purine and Pyrimidine Metabolism Löffler, M., et al., 2005. Pyrimidine pathways in health and disease. Trends in Molecular Medicine 11:430–437. Nyhan, W. L., 2005. Disorders of purine and pyrimidine metabolism. Molecular Genetics and Metabolism 86:25–33. Scriver, C. R., et al., 1995. The Metabolic Bases of Inherited Disease, 7th ed. New York: McGraw-Hill. Metabolic Channeling Benkovic, S. J., 1984. The transformylase enzymes in de novo purine biosynthesis. Trends in Biochemical Sciences 9:320–322. Henikoff, S., 1987. Multifunctional polypeptides for purine de novo synthesis. BioEssays 6:8–13. Huang, X., Holden, H. M., and Raushel, F. M., 2001. Channeling of substrates and intermediates in enzyme-catalyzed reactions. Annual Re- view of Biochemistry 70:149–180. Srere, P. A., 1987. complexes of sequential metabolic enzymes. Annual Review of Biochemistry 56:89–124. Deoxyribonucleotide Biosynthesis Carreras, C. W., and Santi, D. V., 1995. The catalytic mechanism and structure of thymidylate synthase. Annual Review of Biochemistry 64:721–762. Frey, P. A., 2001. Radical mechanisms of enzymatic catalysis. Annual Re- view of Biochemistry 70:121–148. Herrick, J., and Sciavi, B., 2007. Ribonucleotide reductase and the regulation of DNA replication: An old story and an ancient heritage. Molecular Microbiology 63:22–34. Jordan, A., and Reichard, P., 1998. Ribonucleotide reductases. Annual Review of Biochemistry 67:71–98. Licht, S., Gerfen, G. J., and Stubbe, J., 1996. Thiyl radicals in ribonucleotide reductases. Science 271:477–481. Marsh, E. N. G., 1995. A radical approach to enzyme catalysis. BioEssays 17:431–441. Reichard, P., 1988. Interactions between deoxyribonucleotide and DNA synthesis. Annual Review of Biochemistry 57:349–374. Stubbe, J., Ge, J., and Y ee, C. S., 2001. The evolution of ribonucleotide reduction revisited. Trends in Biochemical Sciences 26:93–99. Inhibitors of Purine, Pyrimidine, and Deoxyribonucleotide Biosynthesis as Therapeutic Agents Abeles, R. H., and Alston, T. A., 1990. Enzyme inhibition by fluoro compounds. Journal of Biological Chemistry 265:16705–16708. Galmarini, C. M., Mackey, J. R., and Dumontet, C., 2002. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncology 3:415–424. Hitchings, G. H., 1992. Antagonists of nucleic acid derivatives as medic- inal agents. Annual Review of Pharmacology and Toxicology 32:1–9. Park, B. K., Kitteringham, N. R., and O’Neill, P. M., 2001. Metabolism of fluorine-containing drugs. Annual Review of Pharmacology and Toxi- cology 41:443–470. Zrenner, R., et al., 2006. Pyrimidine and purine biosynthesis and degradation in plants. Annual Review of Plant Biology 57:805–836. Copyright © 2008 Washington Metropolitan Area T ransit Authority 27 Metabolic Integration and Organ Specialization In the preceding chapters, we have explored the major metabolic pathways— glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, photosynthesis, gluconeog enesis, fatty acid oxidation, lipid biosynthesis, amino acid metabolism, and nucleotide metabolism. Several of these pathways are catabolic and serve to generate chemical energy useful to the cell; others are anabolic and use this energy to drive the synthesis of essential biomolecules. Despite their opposing purposes, these reactions typically occur at the same time as nutrient molecules are broken down to provide the building blocks and energy for ongoing biosynthesis. Cells maintain a dynamic steady state through processes that involve considerable metabolic flux. The metabolism that takes place in just a single cell is so complex that it defies detailed quantitative description. However, an appreciation of overall relationships can be achieved by stepping back and considering intermediary metabolism at a systems level of organization. 27.1 Can Systems Analysis Simplify the Complexity of Metabolism? The metabolism of a typical heterotrophic cell can be portrayed by a schematic diagram consisting of just three interconnected functional blocks: (1) catabolism, (2) anabolism, and (3) macromolecular synthesis and growth (Figure 27.1). 1. Catabolism Energy-yielding nutrients are oxidized to CO 2 and H 2 O in catabolism, and most of the electrons liberated are passed to oxygen via an electron- transport pathway coupled to oxidative phosphorylation, resulting in the formation of ATP. Some electrons go to reduce NADP ϩ to NADPH, the source of reducing power for anabolism. Glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, and the pentose phosphate pathway are the principal pathways within this block. The metabolic intermediates in these pathways also serve as substrates for processes within the anabolic block. 2. Anabolism The biosynthetic reactions that form the many cellular molecules collectively comprise anabolism. For thermodynamic reasons, the chemistry of anabolism is more complex than that of catabolism (that is, it takes more energy [and often more steps] to synthesize a molecule than can be produced from its degradation). Metabolic intermediates derived from glycolysis and the citric acid cycle are the precursors for this synthesis, with NADPH supplying the reducing power and ATP the coupling energy. 3. Macromolecular Synthesis and Growth The organic molecules produced in anabolism are the building blocks for creation of macromolecules. Like anabolism, The Washington, D.C., Metro map. The coordinated flow of passengers along different transit lines is an apt metaphor for metabolic regulation. Study of an enzyme, a reaction, or a sequence can be biologically relevant only if its position in the hierarchy of function is kept in mind. Daniel E. Atkinson Cellular Energy Metabolism and Its Regulation (1977) KEY QUESTIONS 27.1 Can Systems Analysis Simplify the Complexity of Metabolism? 27.2 What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism? 27.3 Is There a Good Index of Cellular Energy Status? 27.4 How Is Overall Energy Balance Regulated in Cells? 27.5 How Is Metabolism Integrated in a Multicellular Organism? 27.6 What Regulates Our Eating Behavior? 27.7 Can You Really Live Longer by Eating Less? ESSENTIAL QUESTIONS Cells are systems in a dynamic steady state, maintained by a constant flux of nutrients that serve as energy sources or as raw material for the maintenance of cellular structures. Catabolism and anabolism are ongoing, concomitant processes. What principles underlie the integration of catabolism and energy production with anabolism and energy consumption? How is metabolism integrated in complex organisms with multiple organ systems? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 840 Chapter 27 Metabolic Integration and Organ Specialization macromolecular synthesis is driven by energy from ATP, although indirectly in some cases: GTP is the principal energy source for protein synthesis, CTP for phospholipid synthesis, and UTP for polysaccharide synthesis. However, keep in mind that ATP is the principal phosphoryl donor for formation of GTP, CTP, and UTP from GDP, CDP, and UDP, respectively. Macromolecules are the agents of biological function and information—proteins, nucleic acids, lipids that self-assemble into membranes, and so on. Growth can be represented as cellular accumulation of macromolecules and the partitioning of these materials of function and information into daughter cells in the process of cell division. Only a Few Intermediates Interconnect the Major Metabolic Systems Despite the complexity of processes taking place within each block, the connections between blocks involve only a limited number of substances. Just ten or so kinds of catabolic intermediates from glycolysis, the pentose phosphate pathway, and the citric acid cycle serve as the raw material for most of anabolism: four kinds of sugar phosphates (triose-P, tetrose-P, pentose-P, and hexose-P), three ␣-keto acids (pyruvate, oxaloacetate, and ␣-ketoglutarate), two coenzyme A derivatives (acetyl-CoA and succinyl-CoA), and PEP (phosphoenolpyruvate). ATP and NADPH Couple Anabolism and Catabolism Metabolic intermediates are consumed by anabolic reactions and must be continu- ously replaced by catabolic processes. In contrast, the energy-rich compounds ATP and NADPH are recycled rather than replaced. When these substances are used in Carbohydrates CATABOLISM Fat Protein ANABOLISM Triose-P Tetrose-P Pentose-P Hexose-P PEP Pyruvate Acetyl-CoA ␣-KG Succinyl-CoA Oxaloacetate Amino acids Nucleotides Fatty acids, etc. MACROMOLECULAR SYNTHESIS AND ACCUMULATION (GROWTH) Proteins Nucleic acids Complex lipids Membranes Organelles Cell walls, etc. + PHOTOSYNTHESIS CARBON DIOXIDE FIXATION h␯ ATP ADP NTP NDP NADP + NADPH ATP ADP H 2 O H 2 O O 2 CO 2 CO 2 NADP + NADPH FIGURE 27.1 Block diagram of intermediary metabolism. 27.2 What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism? 841 biosynthesis, the products are ADP and NADP ϩ , and ATP and NADPH are regenerated during catabolism. ATP and NADPH are unique in that they are the only compounds whose purpose is to couple the energy-yielding processes of catabolism to the energy-consuming reactions of anabolism. Certainly, other coupling agents serve essential roles in metabolism. For example, NADH and [FADH 2 ] participate in the transfer of electrons from substrates to O 2 during oxidative phosphorylation. How- ever, these reactions are solely catabolic, and the functions of NADH and [FADH 2 ] are fulfilled within the block called catabolism. Phototrophs Have an Additional Metabolic System— The Photochemical Apparatus The systems in Figure 27.1 reviewed thus far are representative only of metabolism as it exists in aerobic heterotrophs. The photosynthetic production of ATP and NADPH in photoautotrophic organisms entails a fourth block, the photochemical system (Fig- ure 27.1). This block consumes H 2 O and releases O 2 . When this fourth block operates, energy production within the catabolic block can be largely eliminated. Yet another block, one to account for the fixation of carbon dioxide into carbohydrates, is also re- quired for photoautotrophs. The inputs to this fifth block are the products of the photochemical system (ATP and NADPH) and CO 2 derived from the environment. The carbohydrate products of this block may enter catabolism, but not primarily for energy production. In photoautotrophs, carbohydrates are fed into catabolism to generate the metabolic intermediates needed to supply the block of anabolism. Although these diagrams are oversimplifications of the total metabolic processes in heterotrophic or phototrophic cells, they are useful illustrations of functional relationships between the major metabolic subdivisions. This general pattern provides an overall perspective on metabolism, making its purpose easier to understand. 27.2 What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism? Virtually every metabolic pathway either consumes or produces ATP. The amount of ATP involved—that is, the stoichiometry of ATP synthesis or hydrolysis—lies at the heart of metabolic relationships. The overall thermodynamic efficiency of any metabolic sequence, be it catabolic or anabolic, is determined by ATP coupling. In every case, the overall reaction mediated by any metabolic pathway is energetically favorable because of its particular ATP stoichiometry. In the highly exergonic reactions of catabolism, much of the energy released is captured in ATP synthesis. In turn, the thermodynamically unfavorable reactions of anabolism are driven by energy released upon ATP hydrolysis. To illustrate this principle, we must first consider the three types of stoichiometries. The first two are fixed by the laws of chemistry, but the third is unique to living systems and reveals a fundamental difference between the inanimate world of chemistry and physics and the world of biological function, as shaped by evolution— that is, the world of living organisms. The fundamental difference is the stoichiometry of ATP coupling. 1. Reaction Stoichiometry This is simple chemical stoichiometry—the number of each kind of atom in any chemical reaction remains the same, and thus equal num- bers must be present on both sides of the equation. This requirement holds even for a process as complex as cellular respiration: C 6 H 12 O 6 ϩ 6 O 2 ⎯⎯→6 CO 2 ϩ 6 H 2 O The six carbons in glucose appear as 6 CO 2 , the 12 H of glucose appear as the 12 H in six molecules of water, and the 18 oxygens are distributed between CO 2 and H 2 O. 2. Obligate Coupling Stoichiometry Cellular respiration is an oxidation–reduction process, and the oxidation of glucose is coupled to the reduction of NAD ϩ and [FAD]. (Brackets here denote that the relevant FAD is covalently linked to succinate dehy- Stoichiometry is the measurement of the amounts of chemical elements and molecules involved in chemical reactions (from the Greek stoicheion, meaning “element,”and metria, meaning “measure”). 842 Chapter 27 Metabolic Integration and Organ Specialization drogenase; see Chapter 20). The NADH and [FADH 2 ] thus formed are oxidized in the electron-transport pathway: (a) C 6 H 12 O 6 ϩ 10 NAD ϩ ؉ 2 [FAD] ϩ 6 H 2 O ⎯⎯→ 6 CO 2 ϩ 10 NADH ϩ 10 H ϩ ϩ 2 [FADH 2 ] (b) 10 NADH ϩ 10 H ϩ ؉ 2 [FADH 2 ] ϩ 6 O 2 ⎯⎯→12 H 2 O ϩ 10 NAD ϩ ؉ 2 [FAD] Sequence (a) accounts for the oxidation of glucose via glycolysis and the citric acid cycle. Sequence (b) is the overall equation for electron transport per glucose. The stoichiometry of coupling by the biological e Ϫ carriers, NAD ϩ and FAD, is fixed by the chemistry of electron transfer; each of the coenzymes serves as an e Ϫ pair acceptor. Re- duction of each O atom takes an e Ϫ pair. Metabolism must obey these facts of chemistry: Biological oxidation of glucose releases 12 e Ϫ pairs, creating a requirement for 12 equivalents of e Ϫ pair acceptors, which transfer the electrons to 12 O atoms. By evolutionary chance, NAD ϩ /NADH and FAD/FADH 2 carry these electrons, but the stoichiometry is fixed by the chemistry. 3. Evolved Coupling Stoichiometries The participation of ATP is fundamentally different from the role played by pyridine nucleotides and flavins. The stoichiometry of adenine nucleotides in metabolic sequences is not fixed by chemical neces- sity. Instead, the “stoichiometries” we observe are the consequences of evolutionary design. The overall equation for cellular respiration, 1 including the coupled formation of ATP by oxidative phosphorylation, is C 6 H 12 O 6 ϩ 6 O 2 ϩ 38 ADP ϩ 38 P i ⎯⎯→6 CO 2 ϩ 38 ATP ϩ 44 H 2 O The “stoichiometry” of ATP formation, 38 ADP ϩ 38 P i ⎯→38 ATP ϩ 38 H 2 O, cannot be predicted from any chemical considerations. The value of 38 ATP is an end result of biological adaptation. It is a trait that evolved through interactions between chemistry, heredity, and the environment over the course of evolution. Like any evolved character, ATP stoichiometry is the result of compromise. The final trait is one particularly suited to the fitness of the organism. The number 38 is not magical. Recall that in eukaryotes, the consensus value for the net yield of ATP per glucose is 30 to 32, not 38 (see Table 20.4). Also, the value of 38 was established a long time ago in evolution, when the prevailing atmospheric conditions and the competitive situation were undoubtedly very different from those today. The significance of this number is that it provides a high yield of ATP for each glucose molecule, yet the yield is still low enough that essentially all of the glucose is metabolized. ATP Coupling Stoichiometry Determines the K eq for Metabolic Sequences The fundamental biological purpose of ATP as an energy-coupling agent is to drive thermodynamically unfavorable reactions. In effect, the energy release accompa- nying ATP hydrolysis is transmitted to the unfavorable reaction so that the overall free energy change for the coupled process is negative (that is, favorable). The in- volvement of ATP serves to alter the free energy change for a reaction; or to put it another way, the role of ATP is to change the equilibrium ratio of [reactants] to [products] for a reaction. (See the A Deeper Look box on page 67.) Another way of viewing these relationships is to note that, at equilibrium, the concentrations of ADP and P i will be vastly greater than that of ATP because ⌬G°Ј for ATP hydrolysis is a large negative number. 2 However, the cell where this reaction 1 This overall equation for cellular respiration is for the reaction within an uncompartmentalized (prokaryotic) cell. In eukaryotes, where much of the cellular respiration is compartmentalized within mitochondria, mitochondrial ADP/ATP exchange imposes a metabolic cost on the proton gradient of 1 H ϩ per ATP, so the overall yield of ATP per glucose is 32, not 38. 2 Since ⌬G°ЈϭϪ30.5 kJ/mol, ln K eq ϭ 12.3. So K eq ϭ 2.2 ϫ 10 5 . Choosing starting conditions of [ATP] ϭ 8 mM, [ADP] ϭ 8 mM, and [P i ] ϭ 1 mM, we can assume that, at equilibrium, [ATP] has fallen to some insignificant value x, [ADP] ϭ approximately 16 mM, and [P i ] ϭ approximately 9 mM. The concentration of ATP at equilibrium, x, then calculates to be about 1 nM.

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