1. UNIT IV: Nitrogen Metabolism Nucleotide Metabolism Part 2

2. Ribonucleases and deoxyribonucleases, secreted by the pancreas, hydrolyze RNA and DNA primarily to oligonucleotides. Oligonucleotides are further hydrolyzed by pancreatic phosphodiesterases, producing a mixture of 3 ′ - and 5 ′ - mononucleotides. A family of nucleotidases removes the phosphate groups hydrolytically, releasing nucleosides that may be absorbed by the intestinal mucosal cells, or be further degraded to free bases before uptake. Dietary purines and pyrimidines are not used to a large extent for the synthesis of tissue nucleic acids. Instead, the dietary purines are generally converted to uric acid by intestinal mucosal cells. Most of the uric acid enters the blood, and is eventually excreted in the urine. 2 A. Degradation of dietary nucleic acids in the small intestine A. Degradation of dietary nucleic acids in the small intestine

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4. [1] An amino group is removed from AMP to produce IMP, or from adenosine to produce inosine (hypoxanthine-ribose) by AMP deaminase or adenosine deaminase. [2] IMP and GMP are converted into their nucleoside forms— inosine and guanosine—by the action of 5 ′ -nucleotidase. [3] Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine. [4] Guanine is deaminated to form xanthine. [5] Hypoxanthine is oxidized by xanthine oxidase to xanthine, which is further oxidized by xanthine oxidase to uric acid, the final product of human purine degradation. Uric acid is excreted in the urine. 4 B. Formation of uric acid B. Formation of uric acid

5. 5 ADA is expressed in a variety of tissues, but, in humans, lymphocytes have the highest activity of this cytoplasmic enzyme

6. 1. Gout: Gout is a disorder characterized by high levels of uric acid—the end product of purine catabolism—in blood (hyperuricemia), as a result of either the overproduction or underexcretion of uric acid. The hyperuricemia leads to the deposition of monosodium urate crystals in the joints, and an inflammatory response to the crystals, causing first acute and then to chronic gouty arthritis. Nodular masses of monosodium urate crystals (tophi) may be deposited in the soft tissues, resulting in chronic tophaceous gout. 6 C. Diseases associated with purine degradation C. Diseases associated with purine degradation

7. Formation of uric acid stones in the kidney may also be seen. Hyperuricemia is typically asymptomatic and does not lead to gout, but gout is preceded by hyperuricemia The definitive diagnosis of gout requires aspiration and examination of synovial fluid from an affected joint (or material from a tophus) using polarized light microscopy to confirm the presence of needle-shaped monosodium urate crystals 7

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9. Analysis of joint fluid can help to define causes of joint swelling or arthritis, such as infection, gout, and rheumatoid disease 9

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11. Acute attacks of gout are treated with anti-inflammatory agents. Colchicine, steroidal drugs such as prednisone, and nonsteroidal drugs such as indomethacin are used Colchicine depolymerizes microtubules, thus decreasing the movement of neutrophils into the affected area. Like the other anti-inflammatory drugs, it has no effect on uric acid levels. Long-term therapeutic strategies for gout involve lowering the uric acid level below the saturation point, thereby preventing the deposition of urate crystals. 11 C. Treatment of gout C. Treatment of gout

12. Uricosuric agents, such as probenecid or sulfinpyrazone, that increase renal excretion of uric acid, are used in patients who are “underexcretors” of uric acid. Allopurinol, an inhibitor of uric acid synthesis, is used in patients who are “overproducers” of uric acid Allopurinol is converted in the body to oxypurinol, which inhibits xanthine oxidase, resulting in an accumulation of hypoxanthine and xanthine —compounds more soluble than uric acid and, therefore, less likely to initiate an inflammatory response In patients with normal levels of HGPRT, the hypoxanthine can be salvaged, thus reducing the levels of PRPP and, therefore, de novo purine synthesis. 12 C. Treatment of gout C. Treatment of gout

13. VI. Pyrimidine Synthesis and Degradation Unlike the synthesis of the purine ring, which is constructed on a preexisting ribose 5-phosphate, the pyrimidine ring is synthesized before being attached to ribose 5-phosphate, which is donated by PRPP. The sources of the atoms in the pyrimidine ring are glutamine, CO 2, and aspartic acid Therefore, Glutamine and aspartic acid are thus required for both purine and pyrimidine synthesis 13

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15. The regulated step of this pathway in mammalian cells is the synthesis of carbamoyl phosphate from glutamine and CO 2, catalyzed by carbamoyl phosphate synthetase (CPS) II. CPS II is inhibited by UTP (the end product of this pathway, which can be converted into the other pyrimidine nucleotides), and is activated by ATP and PRPP. 15 A. Synthesis of carbamoyl phosphate A. Synthesis of carbamoyl phosphate

16. The second step in pyrimidine synthesis is the formation of carbamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed hydrolytically by dihydroorotase. The resulting dihydroorotate is oxidized to produce orotic acid (orotate). The enzyme that produces orotate, dihydroorotate dehydrogenase, is associated with the inner mitochondrial membrane. All other enzymes in pyrimidine biosynthesis are cytosolic. 16 B. Synthesis of orotic acid B. Synthesis of orotic acid

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18. The completed pyrimidine ring is converted to the nucleotide orotidine 5 ′ -monophosphate (OMP) in the second stage of pyrimidine nucleotide synthesis. PRPP is again the ribose 5-phosphate donor. The enzyme orotate phosphoribosyltransferase produces OMP and releases pyrophosphate, thereby making the reaction biologically irreversible. 18 C. Formation of a pyrimidine nucleotide C. Formation of a pyrimidine nucleotide

19. OMP, the parent pyrimidine mononucleotide, is converted to uridine monophosphate (UMP) by orotidylate decarboxylase, which removes the acidic carboxyl group. Orotate phosphoribosyltransferase and orotidylate decarboxylase are also catalytic domains of a single polypeptide chain called UMP synthase. Orotic aciduria—a rare genetic defect—is caused by a deficiency of this bifunctional enzyme, resulting in orotic acid in the urine (see Figure 22.21). UMP is phosphorylated to UTP. 19 C. Formation of a pyrimidine nucleotide C. Formation of a pyrimidine nucleotide

20. CTP is produced by amination of UTP by CTP synthetase. [Note: The nitrogen is provided by glutamine] 20 D. Synthesis of UTP and cytidine triphosphate (CTP) D. Synthesis of UTP and cytidine triphosphate (CTP)

21. dUMP is converted to dTMP by thymidylate synthase, which uses N5,N10-methylene tetrahydrofolate as the source of the methyl group. This is an unusual reaction in that tetrahydrofolate (THF) contributes not only a one-carbon unit but also two hydrogen atoms from the pteridine ring, resulting in the oxidation of THF to dihydrofolate (DHF). Inhibitors of thymidylate synthase include thymine analogs such as 5-fluorouracil, which serves as successful antitumor agents. 5-Fluorouracil is metabolically converted to 5-FdUMP, which becomes permanently bound to the inactivated thymidylate synthase; for this reason, the drug is called a “suicide” inhibitor. 21 E. Synthesis of thymidine monophosphate (TMP) from dUMP E. Synthesis of thymidine monophosphate (TMP) from dUMP

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23. DHF can be reduced to THF by dihydrofolate reductase, an enzyme that is inhibited in the presence of drugs such as methotrexate. By decreasing the supply of THF, these folate analogs not only inhibit purine synthesis, but, by preventing methylation of dUMP to dTMP, they also lower the cellular concentration of this essential component of DNA. DNA synthesis is inhibited and cell growth slowed. Drugs such as those described above, therefore, are used to decrease the growth rate of cancer cells. 23 E. Synthesis of thymidine monophosphate (TMP) from dUMP E. Synthesis of thymidine monophosphate (TMP) from dUMP

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25. Few pyrimidine bases are salvaged in human cells. However, the pyrimidine nucleosides can be salvaged by nucleoside kinases that utilize ATP in the phosphorylation of the nucleosides to nucleotides. Note: The salvage of pyrimidine nucleosides is the basis for using uridine in the treatment of hereditary orotic aciduria. 25 F. Salvage of pyrimidines F. Salvage of pyrimidines

26. G. Degradation of pyrimidine nucleotides Unlike the purine ring, which is not cleaved by human cells, the pyrimidine ring is opened and degraded to highly soluble products: β -alanine and β -aminoisobutyrate 26 precursors of acetyl CoA and succinyl CoA, respectively), with the production of NH 3 and CO 2.

27. 7. Chapter summary Nucleotides are composed of a nitrogenous base (adenine = A, guanine = G, cytosine = C, uracil = U, and thymine = T), a pentose, and one, two, or three phosphate groups (Figure 22.24). A and G are purines; C, U, and T are pyrimidines. If the sugar is ribose, the nucleotide is a ribonucleoside phosphate (for example, AMP), and it can have several functions in the cell, including being a component of RNA. If the sugar is deoxyribose, the nucleotide is a deoxyribonucleoside phosphate (for example, dAMP), and will be found almost exclusively as a component of DNA. 27

28. 7. Chapter summary The committed step in purine synthesis uses: 5-phosphoribosyl-1-pyrophosphate (PRPP, an “activated pentose” that provides the ribose-phosphate group for de novo purine and pyrimidine synthesis and purine salvage) and nitrogen from glutamine to produce phosphoribosyl amine. The enzyme is glutamine:PRPP amidotransferase, and is inhibited by AMP and GMP (the end products of the pathway) and activated by PRPP. Purine nucleotides can also be produced from preformed purine bases by using salvage reactions catalyzed by adenine phosphoribosyltransferase (APRT) and hypoxanthine- guanine phosphoribosyltransferase (HGPRT). 28

29. 7. Chapter summary A near total deficiency of HGPRT causes Lesch-Nyhan syndrome—a severe, heritable form of hyperuricemia accompanied by compulsive self-mutilation. All deoxyribonucleotides are synthesized from ribonucleotides by the enzyme ribonucleotide reductase. This enzyme is highly regulated, for example, it is strongly inhibited by dATP—a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase deficiency. This syndrome causes severe combined immunodeficiency disease. 29

30. 7. Chapter summary The end product of purine degradation is uric acid—a compound whose overproduction or under-secretion causes hyperuricemia that, if accompanied by the deposition of urate crystals in joints and soft tissues, and an inflammatory response to those crystals, results in gout. The first step in pyrimidine synthesis—the production of carbamoyl phosphate by carbamoyl phosphate synthetase II—is the regulated step in this pathway (it is inhibited by UTP and activated by PRPP). 30

31. 7. Chapter summary The UTP produced by this pathway can be converted to CTP. dUMP can be converted to dTMP using thymidylate synthase—an enzyme targeted by anticancer drugs such as 5-fluorouracil. The regeneration of THF from DHF produced in the thymidylate synthase reaction requires dihydrofolate reductase—an enzyme targeted by the drug, methotrexate. 31

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