Chapter 25

1. In de novo synthesis, the nucleotides are synthesized from simpler precursor compounds, in essence from scratch. In salvage pathways, preformed bases are recovered and attached to riboses.

2. Carbon 2 and nitrogen 3 come from carbamoyl phosphate. Nitrogen 1 and carbons 4, 5, and 6 are derived from aspartate.

3. Nitrogen 1: aspartate; carbon 2: N10-formyltetrahydrofolate; nitrogen 3: glutamine; carbons 4 and 5 and nitrogen 7: glycine; carbon 6: CO2; carbon 8: N10-formyltetrahydrofolate; nitrogen 9: glutamine.

4. Energy currency: ATP; signal transduction: ATP and GTP; RNA synthesis: ATP, GTP, CTP, and UTP; DNA synthesis: dATP, dCTP, dGTP, and TTP; components of coenzymes: ATP in CoA, FAD, and NAD(P)+; carbohydrate synthesis: UDP-glucose. These are just some of the uses.

5. A nucleoside is a base attached to ribose. A nucleotide is a nucleoside with the ribose bearing one or more phosphates.

6. (a) 9; (b) 7; (c) 6; (d) 10; (e) 2; (f ) 4; (g) 1; (h) 11; (i) 8; ( j) 3; (k) 5.

7. Substrate channeling is the process whereby the product of one active site moves to become a substrate at another active site without ever leaving the enzyme. A channel connects the active sites. Substrate channeling greatly enhances enzyme efficiency and minimizes the diffusion of a substrate to an active site.

8. Glucose + 2 ATP + 2 NADP+ + 1 H2O → PRPP + CO2 + ADP + AMP + 2 NADPH + 3 H+.

9. Glutamine + aspartate + CO2 + 2 ATP + NAD+ → orotate + 2 ADP + 2 Pi + glutamate + NADH + H+.

10. (a, c, and d) PRPP; (b) carbamoyl phosphate.

11. PRPP and formylglycinamide ribonucleotide

12. dUMP + serine + NADPH +H+ → TMP + NADP+ + glycine.

13. There is a deficiency of N10-formyltetrahydrofolate.

Sulfanilamide inhibits the synthesis of folate by acting as an analog of p-aminobenzoate, one of the precursors of folate.

14. (a) Cell A cannot grow in a HAT medium, because it cannot synthesize TMP either from thymidine or from dUMP. Cell B cannot grow in this medium, because it cannot synthesize purines by either the de novo pathway or the salvage pathway. Cell C can grow in a HAT medium because it contains active thymidine kinase from cell B (enabling it to phosphorylate thymidine to TMP) and hypoxanthine guanine phosphoribosyltransferase from cell A (enabling it to synthesize purines from hypoxanthine by the salvage pathway).

(b) Transform cell A with a plasmid containing foreign genes of interest and a functional thymidine kinase gene. The only cells that will grow in a HAT medium are those that have acquired a thymidylate kinase gene; nearly all of these transformed cells will also contain the other genes on the plasmid.

15. The folate derivative N5, N10-methylenetetrahydrofolate is required by thymidylate synthase to add a methyl group to dUMP, forming TMP. Insufficient folate could result in spina bifida.

16. The reciprocal substrate relation refers to the fact that AMP synthesis requires GTP, whereas GMP synthesis requires ATP. These requirements tend to balance the synthesis of ATP and GTP.

17. Ring carbon 6 in cytosine will be labeled. In guanine, only carbon 5 will be labeled with 13C.

18. UTP is first converted into UDP. Ribonucleotide reductase generates dUDP. DeoxyUDP is converted to dUMP. Thymidylate synthase generates TMP from dUMP. Monophosphate and diphosphate kinases subsequently form TTP.

19. These patients have a high level of urate because of the breakdown of nucleic acids. Allopurinol prevents the formation of kidney stones and blocks other deleterious consequences of hyperuricemia by preventing the formation of urate.

20. The free energies of binding are −57.7 (wild type), −49.8 (Asn 27), and −38.1 (Ser 27) kJ mol−1 (−13.8, −11.9, and −9.1 kcal mol−1, respectively). The loss in binding energy is +7.9 kJ mol−1 (+1.9 kcal mol−1) and +19.7 kJ mol−1 (+4.7 kcal mol−1).

21. By their nature, cancer cells divide rapidly and thus require frequent DNA synthesis. Inhibitors of TMP synthesis will impair DNA synthesis and cancer growth.

22. Uridine and cytidine are administered to by-pass the defective enzyme in orotic aciduria.

23. Inosine or hypoxanthine could be administered.

24. N-1 in both cases, and the amine group linked to C-6 in ATP.

25. Nitrogen atoms 3 and 9 in the purine ring

26. Allopurinol, an analog of hypoxanthine, is a suicide inhibitor of xanthine oxidase.

27. An oxygen atom is added to allopurinol to form alloxanthine.

28.

The synthesis of carbamoyl phosphate requires 2 ATP

2 ATP

The formation of PRPP from ribose 5-phosphate yields an AMP*

2 ATP

The conversion of UMP to UTP requires 2 ATP

2 ATP

The conversion of UTP to CTP requires 1 ATP

1 ATP

Total

7 ATP

*Remember that AMP is the equivalent of 2 ATP because an ATP must be expended to generate ADP, the substrate for ATP synthesis.

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29. (a) Carboxyaminoimidazole ribonucleotide; (b) glycinamide ribonucleotide; (c) phosphoribosyl amine; (d) formylglycinamide ribonucleotide.

30. The first reaction proceeds by phosphorylation of glycine to form an acyl phosphate followed by nucleophilic attack by the amine of phosphoribosylamine to displace orthophosphate. The second reaction consists of adenylation of the carbonyl group of xanthylate followed by nucleophilic attack by ammonia to displace AMP.

31. The −NH2 group attacks the carbonyl carbon atom to form a tetrahedral intermediate. Removal of a proton leads to the elimination of water to form inosinate.

32. The enzyme that uses ammonia, carbamoyl phosphate synthetase I, forms carbamoyl phosphate for a reaction with ornithine, the first step of the urea cycle. The enzyme that uses glutamine, carbamoyl phosphate synthetase II, generates carbamoyl phosphate for use in the first step of pyrimidine biosynthesis.

33. PRPP is the activated intermediate in the synthesis of phosphoribosylamine in the de novo pathway of purine formation; of purine nucleotides from free bases by the salvage pathway; of orotidylate in the formation of pyrimidines; of nicotinate ribonucleotide; of phosphoribosyl ATP in the pathway leading to histidine; and of phosphoribosylanthranilate in the pathway leading to tryptophan.

34. (a) cAMP; (b) ATP; (c) UDP-glucose; (d) acetyl CoA; (e) NAD+, FAD; (f) dideoxynucleotides; (g) fluorouracil; (h) CTP inhibits ATCase.

35. In vitamin B12 deficiency, methyltetrahydrofolate cannot donate its methyl group to homocysteine to regenerate methionine. Because the synthesis of methyltetrahydrofolate is irreversible, the cell’s tetrahydrofolate will ultimately be converted into this form. No formyl or methylene tetrahydrofolate will be left for nucleotide synthesis. Vitamin B12 is also required to metabolize propionyl CoA generated in the oxidation of odd-chain fatty acids and in the degradation of methionine.

36. Because folate is required for nucleotide synthesis, cells that are dividing rapidly would be most readily affected. They would include cells of the intestine, which are constantly replaced, and precursors to blood cells. A lack of intestinal cells and blood cells would account for the symptoms often observed.

37. In patients with glucose 6-phosphatase deficiency, the cytoplasmic level of ATP in the liver falls as a result of increased glycogenolysis. In all three conditions, AMP rises above normal, and the excess AMP is degraded to urate.

38. Succinate → malate → oxaloacetate by the citric acid cycle. Oxaloacetate → aspartate by transamination, followed by pyrimidine synthesis. Carbons 4, 5, and 6 are labeled.

39. Glucose will most likely be converted into two molecules of pyruvate, one of which will be labeled in the 2 position:

Now consider two common fates of pyruvate—conversion into acetyl CoA and subsequent processing by the citric acid cycle or carboxylation by pyruvate carboxylase to form oxaloacetate. Formation of citrate by condensing the labeled pyruvate with oxaloacetate will yield labeled citrate:

The labeled carbon will be retained through one round of the citric acid cycle but, on the formation of the symmetric succinate, the label will appear in two different positions. Thus, when succinate is metabolized to oxaloacetate, which may be aminated to form aspartate, two carbons will be labeled:

When this aspartate is used to form uracil, the labeled COO attached to the α-carbon is lost and the other COO becomes incorporated into uracil as carbon 4.

Suppose, instead, that labeled 2-[14C]pyruvate is carboxylated to form oxaloacetate and processed to form aspartate. In this case, the α-carbon of aspartate bears the label.

When this aspartate is used to synthesize uracil, carbon 6 bears the label:

40. HGPRT, which is nonfunctional in Lesch–Nyhan patients, is required to form 6-mercaptopurine ribose monophosphate. Consequently, de novo purine synthesis continues.

41. (a) Some ATP can be salvaged from the ADP that is being generated. (b) There are equal numbers of high-phosphoryl-transfer-potential groups on each side of the equation. (c) Because the adenylate kinase reaction is at equilibrium, the removal of AMP would lead to the formation of more ATP. (d) Essentially, the cycle serves as an anaplerotic reaction for the generation of the citric acid cycle intermediate fumarate.

42. (i) The formation of 5-aminoididazole-4-carboxamide ribonucleotide from 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide in the synthesis of IMP. (ii) The formation of AMP from adenylosuccinate. (iii) The formation of arginine from argininosuccinate in the urea cycle.

43. Allopurinol is an inhibitor of xanthine oxidase, which is on the pathway for urate synthesis. In your pet duck, this pathway is the means by which excess nitrogen is excreted. If xanthine oxidase were inhibited in your duck, nitrogen could not be excreted, with severe consequences such as the formation of a dead duck.

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44. (a) The enzyme from the LND 2 patient showed much less activity than the normal enzyme, suggesting that the defect in the enzyme impaired its catalytic ability. The results for LND 1 are puzzling. The enzyme displays activity similar to the enzyme from the normal cell line, and yet the patient was suffering from Lesch–Nyhan disease.

(b) Possible explanations include: there may be an inhibitor in the cells that prevents the enzyme from acting in vivo but is lost during the purification procedure; the enzyme may be degraded more rapidly in vivo than the normal enzyme; the enzyme may be inherently less stable than the normal enzyme.

(c) The enzyme from LND 1 lost activity much faster than the normal enzyme, suggesting that the enzyme was structurally unstable and would lose enzyme activity in the cell, accounting for the appearance of the disease.