Chapter 28: Nucleotide metabolism
Quiz on Mon (4/16): IMP synthesis-Purine synthesis
Quiz on Wed(4/18): Pyrimidine biosynthesis/regulation
Quiz on Friday(4/20): Ribonucleotide reductase mechanism
Friday (4/20): extra credit seminar, Dr. Jimmy Hougland,
145 Baker, 3-4PM.
ACS exam has been moved to Monday (4/30)
Quiz on Final is scheduled for May 4, 12:45PM-2:45PM, in 111 Marshall

• Synthesis of pyrimidine nucleotide triphosphates is similar to purine nucleotide triphosphates.
• 2 sequential enzymatic reactions catalyzed by nucleoside monophosphate kinase and nucleoside diphosphate kinase respectively:
UMP + ATP

UDP + ADP
UDP + ATP

UTP + ADP

CTP is formed by amination of UTP by CTP synthetase
In animals, amino group from Gln
In bacteria, amino group from ammonia

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Bacteria regulated at Reaction 2 (ATCase)
Allosteric activation by ATP
Inhibition by CTP (in E. coli ) or UTP (in other bacteria).
In animals pyrimidine biosynthesis is controled by carbamoyl phosphate synthetase II
Inhibited by UDP and UTP
Activated by ATP and PRPP
Mammals have a second control at OMP decarboxylase (competitively inhibited by
UMP and CMP)
PRPP also affects rate of OMP production, so, ADP and GDP will inhibit PRPP production.

• Derived from corresponding ribonucleotides by reduction of the C2’ position.
• Catalyzed by ribonucleotide reductases (RNRs)
ADP dADP

Overview of dNTP biosynthesis
One enzyme, ribonucleotide reductase, reduces all four ribonucleotides to their deoxyribose derivatives.
A free radical mechanism is involved in the ribonucleotide reductase reaction.
There are three classes of ribonucleotide reductase enzymes in nature:
Class I: tyrosine radical, uses NDP
Class II: adenosylcobalamin. uses NTPs
(cyanobacteria, some bacteria,
Euglena).
Class III: SAM and Fe-S to generate radical, uses NTPs.
(anaerobes and fac. anaerobes).

Figure 28-12a Class I ribonucleotide reductase from E. coli . ( a ) A schematic diagram of its quaternary structure.

Proposed mechanism for rNDP reductase

Proposed reaction mechanism for ribonucleotide reductase
1.
Free radical abstracts H from
C3’
2.
Acid-catalyzed cleavage of the
C2’-OH bond
3.
Radical mediates stabilizationof the
C2’ cation
(unshared electron pair)
4.
Radical-cation intermediate is reduced by redoxactive sulhydryl pairdeoxynucleotide radical
5.
3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

• Final step in the RNR catalytic cycle is the reduction of disulfide bond to reform the redox-active sulfyhydryl pair).
• Thioredoxin-108 residue protein that has redox active Cys (Cys32 and
Cys35)-also involved in the Calvin Cycle.
• Reduces oxidized RNR and is regenerated via NADPH by thioredoxin reductase.
• Glutaredoxin is an 85 residue protein that can also reduce RNR.
• Oxidized glutaredoxin is reuced by NADPH using glutredeoxin reductase.

Sources of reducing power for rNDP reductase

Proposed reaction mechanism for ribonucleotide reductase
1.
Free radical abstracts H from
C3’
2.
Acid-catalyzed cleavage of the
C2’-OH bond
3.
Radical mediates stabilizationof the
C2’ cation
(unshared electron pair)
4.
Radical-cation intermediate is reduced by redoxactive sulhydryl pairdeoxynucleotide radical
5.
3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

• Reaction is catalyzed by nucleoside diphosphate kinase (same enzyme that phosphorylates NDPs) dNDP + ATP
 dNTP + ADP
• Can use any NTP or dNTP as phosphoryl donor.

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2 main enzymes: dUTP diphosphohydrolase (dUTPase) and thymidylate synthase
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Reaction 1 dTMP is made by methylation of dUMP.
dUMP is made by hydrolysis of dUTP via dUTP diphosphohydrolase (dUTPase) dUTP + H
2
O
 dUMP+ PP i
Done to minimize the concentration of dUTP-prevents incorporation of uracil into DNA.

Reaction 2
• dTMP is made from dUMP by thymidylate synthase (TS).
• Uses N5, N10-methylene-THF as methyl donor
+ dUMP
+ dTMP

Figure 28-19 Catalytic mechanism of thymidylate synthase.
1.
Enzyme Cys thiolate group attacks C6 of dUMP (nucleophile).
2.
C5 of the enolate ion attacks the CH
2 group of the imium cation of N 5 , N 10 -methylene-
THF.
3.
Enzyme base abstracts the acidic proton at
C5, forms methylene group and eliminates
THF cofactor
4.
Migration of the N6-H atom of THF to the exocyclic methylene group to form a methyl group and displace the Cys thiolate intermediate.

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Antitumor agent.
Irreversible inhibitor of TS
Binds like dUMP but in step 3 of the reaction, F cannot be extracted.
Suicide substrate.
F
FdUMP

Figure 28-20 The X-ray structure of the E. coli thymidylate synthase –FdUMP–THF ternary complex.

N 5
N 10
• Only enzyme to change the oxidation state of THF.
• Regenerated by 2 reactions
• DHF is reduced to THF by NADPH by dihydrofolate reductase.
• Serine hydroxymethyltransferase transfers the hydroxymethyl group of serine to THF to regenerate N 5 , N 10 methylene-THF and produces glycine.

Figure 28-21 Regeneration of N 5, N 10methylenetetrahydrofolate.

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Nucleic acids can survive the acid of the stomach
Degraded into nucleotides by pancreatic nucleases and intestinal phosphodiesterases in the duodenum.
Components cannot pass through cell membranes, so they are hydrolyzed to nucleosides.
Nucleosides may be directly absorbed by the intestine or undergo further degradation to free bases and ribose or ribose-1-phosphate by nucleosidases and nucloside phosphorylase .
Nucleoside + H
2
O base + ribose nucleosidase
Nucleoside + P i base + ribose-1-P
Nucleoside phosphorylase

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All pathways lead to formation of uric acid.
Intermediates could be intercepted into salvage pathways.
1st reaction is the nucleotidase and second is catalyzed by purine nucleoside phosphorylase (PNP)
Ribose-1-phosphate is isomerized by phosphoribomutase to ribose-5-phosphate
(precursor to PRPP).
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Purine nucleoside + P i
Purine base + ribose-1-P
Adenosine and deoxyadenosine are not degraded by PNP but are deaminated by adenosine deaminase (ADA) and AMP deaminase in mammals

Figure 28-23 Major pathways of purine catabolism in animals.
ADA
Genetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese
(SCID).
No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPs

Figure 28-24a Structure and mechanism of adenosine deaminase. ( a ) A ribbon diagram of murine adenosine deaminase in complex with its transition state analog HDPR.

Figure 28-24b ( b ) The proposed catalytic mechanism of adenosine deaminase.
1.
Zn 2+ polarized H
2
O molecule nucleophilically attacks C6 of the adenosine. His is general base catalyst,
Glu is general acid, and Asp orients water.
2.
Results in tetrahedral intermediate which decomposes by elimination of ammonia.
3.
Product is inosine in enol form (assumes dominant keto form upon release from enzyme).

• Deamination of AMP to IMP combined with synthesis of AMP from IMP results in deaminating Asp to yield fumarate.
• Important role in skeletal muscle-increased activity requires increased activity in the citric acid cycle.
• Muscle replenishes citric acid cycle intermediates through the purine nucleotide cycle.

Figure 28-25 The purine nucleotide cycle.

• Xanthine oxidse (XO) converts hypoxanthine to xanthine, and xanthine to uric acid.
• In mammals, found in the liver and small intestine mucosa
• XO is a homodimer with FAD, two [2Fe-2S] clusters and a molybdopterin complex (Mo-pt) that cycles between Mol (VI) and Mol (IV) oxidation states.
• Final electron acceptor is O
2 which is converted to H
2
O
2
• XO is cleaved into 3 segments. The uncleaved enzyme is known as xanthine dehydrogenase (uses NAD+ as an electron acceptor where XO does not).
• XO hydroxylates hypoxanthine at its C2 position and xanthine at the C8 positon to produce uric acid in the enol form.

Figure 28-26a X-Ray structure of xanthine oxidase from cow’s milk in complex with salicylic acid.
N-terminal domain is cyan
Central domain is gold
C-terminal domain is lavender

1.
Reaction initiated by attack of enzyme nucleophile on the C8 position of xanthine.
2.
The C8-H atom is eliminated as a hydride ion that combines with Mo (VI) complex, reducing it to Mo (IV).
3.
Water displaces the enzyme nucleophile producing uric acid.

Figure 28-27 Mechanism of xanthine oxidase.

Figure 28-23 Major pathways of purine catabolism in animals.
ADA
Genetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese
(SCID).
No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPs

Purine degredation in other animals
Primates, birds, reptiles, insects-final degradation product id uric acid which is excreted in urine.
Goal is the conservation of water.

Figure 28-29 The Gout , a cartoon by James Gilroy
(1799).
Gout is a disease characterized by elevated levels of uric acid in body fluids. Caused by deposition of nearly insoluble crystals of sodium urate or uric acid.

Clinical disorders of purine metabolism
Excessive accumulation of uric acid: Gout
The three defects shown each result in elevated de novo purine biosynthesis

Common treatment for gout: allopurinol
Allopurinol is an analogue of hypoxanthine that strongly inhibits xanthine oxidase. Xanthine and hypoxanthine, which are soluble, are accumulated and excreted.

• Animal cells degrade pyrimidines to their component bases.
• Happen through dephosphorylation, deamination, and glycosidic bond cleavage.
• Uracil and thymine broken down by reduction (vs. oxidation in purine catabolism).

Biosynthesis of of
NAD and NADP+
Produced from vitamin precursors Nicotinate and Nicotinamide and from quinolinate, a Trp degradation product

Biosynthesis of FMN and
FAD from riboflavin
FAD is synthesized from riboflavin in a tworeaction pathway.
Flavokinase phosphorylates the 5’OH group to give FMN
FAD pyrophosphorylase catalyzes the next step
(coupling of FMN to
ADP).

Biosynthesis of CoA from pantothenate