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February 19 Chapter 27 Nucleic acid metabolism

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1 February 19 Chapter 27 Nucleic acid metabolism
Biochemistry 432/832 February 19 Chapter 27 Nucleic acid metabolism

2 Announcements: -

3 Outline 27.1 Nucleotide Biosynthesis 27.2 The Biosynthesis of Purines
27.3 Purine Salvage 27.4 Purine Degradation 27.5 Biosynthesis of Pyrimidines 27.6 Pyrimidine Degradation 27.7 Deoxyribonucleotide Biosynthesis 27.8 Synthesis of Thymine Nucleotides

4 Nucleotides: - Participate in the majority of biochemical reactions - ATP - - energy currency (also ADP, AMP) - UDP-glucose - - glycogen biosynthesis - CoA, NAD+, NADP+, FAD are derivatives of nucleotides - cAMP - - regulation of cellular processes (signaling) - Nucleoside triphosphates (NTP) - - RNA - Deoxynucleoside triphosphates (dNTP) - - DNA

5 Nitrogenous bases

6 Nitrogenous Bases Pyrimidines Purines Cytosine (DNA, RNA) Uracil (RNA)
Thymine (DNA) Purines Adenine (DNA, RNA) Guanine (DNA, RNA)

7 The common bases The common pyrimidine bases The common purine bases

8 The common ribonucleosides

9 Nucleotide biosynthesis:
- Most organisms can make purine and pyrimidine nucleotides via de novo (from scratch) pathways - They can also recover nucleotides from diet - Rapidly dividing cells require large amounts of RNA and DNA - In these cells, large quantities of nucleotides are needed - These pathways are attracting targets for treatment of cancer and infectious microorganisms - Many antibiotics and anticancer drugs are inhibitors of nucleotide biosynthesis

10 Nucleotide biosynthesis: Principles and differences
- Purines: Successive addition of atoms Ribose-5-phosphate serves as a base for the addition of successive atoms derived from common metabolic intermediates - Pyrimidines: Synthesized directly from two common metabolic intermediates Nucleotides are synthesized prior to their linkage to ribose-5-phosphate

11 Nucleotide biosynthesis: Purine biosynthesis
- Elucidated by using isotope labeled compounds - Nine atoms of purine ring are formed by: N1 aspartic acid N3, N9 glutamine C4, C5, N7 glycine C6 carbon dioxide C2, C8 THF derivatives - Ribose-5-phosphate is the initial substrate - Atoms are successively added to R-5-P

12 The metabolic origin of the nine atoms in the purine ring system

13 Inosine-5'-P Biosynthesis
The purine ring is built on a ribose-5-P foundation First step: ribose-5-P must be activated - by PPi 5-phosphoribosyl-a-pyrophosphate (PRPP) is limiting substance for purine synthesis But PRPP is a branch point so the next step is the committed step - Gln PRPP amidotransferase Azaserine - Gln analog - inhibitor/anti-tumor 6 steps use ATP, but that this is really seven ATP equivalents IMP is the immediate precursor of GMP and AMP

14 The pathway for purine biosynthesis

15 Azaserine is an irreversible inhibitor of glutamine-dependent enzymes

16 Reciprocal control occurs in two ways
Making AMP and GMP Reciprocal control occurs in two ways GTP is the energy input for AMP synthesis, whereas ATP is the energy input for GMP AMP is made by N addition from aspartate GMP is made by oxidation at C-2, followed by replacement of the O by N (from Gln)

17 The synthesis of AMP and GMP from IMP

18 Regulation of purine biosynthesis

19 Purine catabolism leads to uric acid
Purine Degradation Purine catabolism leads to uric acid Nucleotidases and nucleosidases release ribose and phosphates and leave free bases Xanthine oxidase and guanine deaminase route everything to xanthine Xanthine oxidase converts xanthine to uric acid Xanthine oxidase can oxidize two different sites on the purine ring system

20 Purine catabolism in animals

21 Xanthine Oxidase and Gout
XO in liver, intestines (and milk) can oxidize hypoxanthine (twice) to uric acid Humans and other primates excrete uric acid in the urine, but most N goes out as urea Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound Gout occurs from accumulation of uric acid crystals in the extremities Allopurinol, which inhibits XO, is a treatment

22 Inhibition of xanthine oxidase

23 The xanthine oxidase reaction

24 Pyrimidine Biosynthesis
In contrast to purines, pyrimidines are not synthesized as nucleotides Rather, the pyrimidine ring is completed before a ribose-5-P is added Carbamoyl-P and aspartate are the precursors of the six atoms of the pyrimidine ring

25 The metabolic origin of the six atoms of the pyrimidine ring

26 CPS II Carbamoyl phosphate for pyrimidine synthesis is made by carbamoyl phosphate synthetase II (CPS II) This is a cytosolic enzyme (whereas CPS I is mitochondrial and used for the urea cycle) Substrates are HCO3-, glutamine, 2 ATP

27 Carbamoyl phosphate synthetase II reaction

28 The pyrimidine biosynthetic pathway

29 Metabolic channeling Eukaryotic pyrimidine synthesis involves substrate channeling and multifunctional polypeptides Advantages: the product of one reaction is the substrate for the next; substrates are not diluted, intermediates do not accumulate; kinetic advantages

30 UDP is made from UMP, and UTP is made from UDP
CTP sythetase forms CTP from UTP (and ATP as energy and glutamine as N source)

31 CTP synthesis from UTP

32 Control of pyrimidine biosynthesis in bacteria and animals

33 Pyrimidine degradation

34 DNA synthesis Synthesis of deoxyribo-nucleotides --- reduction at the 2’-position of the ribose ring of nucleoside diphosphates

35 Deoxyribonucleotide Biosynthesis
Reduction at 2’-position commits nucleotides to DNA synthesis Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase An 22-type enzyme - subunits R1 (86 kDa) and R2 (43.5 kDa) R1 has two regulatory sites, a specificity site and an overall activity site

36 Ribonucleotide Reductase
Activity depends on Cys439, Cys225, and Cys462 on R1 and on Tyr122 on R2 Cys439 removes 3’-H, and dehydration follows, with disulfide formation between Cys225 and Cys462 The net result is hydride transfer to C-2’ Thioredoxin and thioredoxin reductase deliver reducing equivalents

37 E.coli ribonucleotide reductase

38 The free radical mechanism of ribonucleotide reduction

39 Electron transfer from NADPH to RR

40 Glutathione (GSH)

41 Glutathione and thioredoxin reduction

42 Substitution of the thioredoxin system for glutathione reductase in Drosophila
Kanzok et al., Science 291, , 26 January 2001 NADPH + TrxS2 + H+ --> NADP+ + Trx(SH)2 Trx(SH)2 + GSSG --> TrxS2 + 2GSH


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