- As noted previ- ously, these two regulatory sites are designated the overall activity site and the sub- strate specificity site. - ATP is an allosteric activator and dATP is an allosteric inhibitor, 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. - The ratio- nale for these varying affinities is as follows (Figure 26.23): High [ATP] is consis- tent 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. - 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 sub- strates, activators, and inhibitors are such that the formation of dNDPs proceeds in an orderly and balanced fashion. - 26.8 How Are Thymine Nucleotides Synthesized?. - FIGURE 26.23 Regulation of deoxynucleotide biosyn- thesis: the rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribo- nucleotide reductase.. - dCDP can lead to formation of dUMP, the immediate precursor for dTMP syn- thesis (Figure 26.24). - dTMP dTMP FIGURE 26.24 Pathways of dTMP synthesis. - 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 F O C 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. - FCH 2 COO is exceptionally toxic because it is read- ily converted to fluorocitrate by citrate synthase of the citric acid cycle (see Chapter 19). - A thiol group on this enzyme normally attacks the 6-position of the uracil moiety of 2 -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 and dissociation of product dTMP. - Enzyme in- hibitors 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.. - 䊳 The effect of the 5-fluoro substitution on the mechanism of action of thymidylate synthase. - Normally, free enzyme is regenerated following release of the hydrogen at C-5 as a proton.. - dCDP, which is dephosphorylated to dCMP and then deaminated by dCMP deaminase (Figure 26.25), leaving dUMP. - Of the four dNTPs, only dCTP does not interact with either of the regulatory sites on ribonucleotide reductase (see Fig- ure 26.20). - Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase (Figure 26.26). - FIGURE 26.25 (a) The dCMP deaminase reaction.(b) Trimeric dCMP deaminase. - see part a of the figure) is a thymine analog. - 5-FU is used as a chemotherapeutic agent in the treatment of human cancers. - 26.1 Can Cells Synthesize Nucleotides? Nucleotides are ubiquitous constituents of life and nearly all cells are capable of synthesizing them. - 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. - one leading to AMP and the other to GMP. - 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. - 26.3 Can Cells Salvage Purines? Purine ring systems represent a meta- bolic 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). - Thymidylate synthase. - FIGURE 26.26 (a) The thymidylate synthase reaction. - Draw the purine and pyrimidine ring structures, indicating the metabolic source of each atom in the rings.. - 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?. - Illustrate the key points of regulation in (a) the biosynthesis of IMP, AMP, and GMP. - (Integrates with Chapter 25.) At which steps does the purine biosyn- thetic pathway resemble the pathway for biosynthesis of the amino acid histidine?. - Write a balanced equation for the conversion of aspartate to fu- marate by the purine nucleoside cycle in skeletal muscle.. - 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.. - In analogy with the behavior of glycogen phosphorylase shown in Figure 15.14, il- lustrate 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.. - (Integrates with Chapter 20.) Starting from HCO 3 , glutamine, as- partate, 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 biosynthe- sis calculated in problem 2?. - Write a balanced equation for the synthesis of dTMP from UMP and N 5 ,N 10 -methylene-THF. - Postulate a role for the side chains of these Arg residues.. - coli dihydrofolate reductase (DFR) with NADP and folate bound can be found in the Protein Data Bank thine to form IMP or guanine to form GMP. - 26.4 How Are Purines Degraded? Dietary nucleic acids are digested to nucleotides by various nucleases and phosphodiesterases, the nu- cleotides are converted to nucleosides by base-specific nucleotidases and nonspecific phosphatases, and then nucleosides are hydrolyzed to release the purine base. - 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. - 26.6 How Are Pyrimidines Degraded? Degradation of the pyrimi- dine ring generates -alanine, CO 2 , and ammonia. - 26.7 How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? 2-Deoxyribonucleotides are formed from ribonu- cleotides through reduction at the 2-position of the ribose ring in NDPs.. - 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 sub- strates, 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. - in the search line, and click on “KiNG” under “Display options”. - 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 and the pterin ring of folate. - 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). - 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. - Interpret what you see in terms of the Monod–. - Preparing for the MCAT Exam. - 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.. - Decide from Figures 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.. - Kisker, C., Schindelin, H., and Rees, D. - Evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli. - Ribonucleotide reductase and the reg- ulation of DNA replication: An old story and an ancient heritage.. - Jordan, A., and Reichard, P., 1998. - Stubbe, J., Ge, J., and Yee, C. - In the preceding chapters, we have explored the major metabolic pathways—. - Catabolism Energy-yielding nutrients are oxidized to CO 2 and H 2 O in catabo- lism, and most of the electrons liberated are passed to oxygen via an electron- transport pathway coupled to oxidative phosphorylation, resulting in the forma- tion of ATP. - Glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, and the pentose phosphate pathway are the prin- cipal pathways within this block. - 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.. - 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.. - 27.2 What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism?. - 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.. - Just ten or so kinds of catabolic intermediates from glycolysis, the pentose phosphate pathway, and the cit- ric 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 (pyru- vate, oxaloacetate, and -ketoglutarate), two coenzyme A derivatives (acetyl-CoA and succinyl-CoA), and PEP (phosphoenolpyruvate).. - 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.. - The inputs to this fifth block are the products of the pho- tochemical system (ATP and NADPH) and CO 2 derived from the environment. - 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. - In the highly exergonic reactions of catabolism, much of the energy released is captured in ATP synthesis. - 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—. - The fundamental difference is the stoichiome- try of ATP coupling.. - 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. - 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.. - Obligate Coupling Stoichiometry Cellular respiration is an oxidation–reduction process, and the oxidation of glucose is coupled to the reduction of NAD and [FAD].. - Stoichiometry is the measurement of the amounts of chemical elements and molecules involved in chemical reactions (from the Greek stoicheion, meaning “element,” and metria, mean- ing “measure”).. - The NADH and [FADH 2 ] thus formed are oxidized in the electron-transport pathway:. - Sequence (a) accounts for the oxidation of glucose via glycolysis and the citric acid cycle. - each of the coenzymes serves as an e pair acceptor. - The overall equation for cellular respiration, 1 including the coupled for- mation of ATP by oxidative phosphorylation, is. - It is a trait that evolved through interactions between chemistry, heredity, and the environment over the course of evolution.. - The final trait is one particularly suited to the fitness of the organism.. - 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.. - 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). - 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 per ATP, so the overall yield of ATP per glucose is 32, not 38.
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