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FCH 532 Lecture 27
Chapter 28: Nucleotide metabolism
Quiz on Monday (4/18) - IMP biosynthesis
pathway
ACS exam has been moved to Monday (5/2)
Final is scheduled for May 11, 8-10AM, in 111
Marshall
Page 1071
Figure 28-5
Control
network for the
purine biosynthesis
pathway.
Page 1075
Feedback
inhibition is
indicated by
red arrows
Feedforward
activation by
green arrows.
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).
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.
Thioredoxin and glutaredoxin
•
•
•
•
•
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.
dNTPs made by phosphorylation of dNDP
•
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.
Thymine synthesis
•
2 main enzymes: dUTP diphosphohydrolase (dUTPase) and
thymidylate synthase
Reaction 1
•
dTMP is made by methylation of dUMP.
•
dUMP is made by hydrolysis of dUTP via dUTP diphosphohydrolase
(dUTPase)
dUTP + H2O  dUMP+ PPi
•
Done to minimize the concentration of dUTP-prevents incorporation of
uracil into DNA.
Thymine synthesis
Reaction 2
•
dTMP is made from dUMP by thymidylate synthase (TS).
•
Uses N5, N10-methylene-THF as methyl donor
+
dUMP
+
dTMP
Page 1090
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 CH2 group
of the imium cation of N5, N10-methyleneTHF.
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.
5-flurodeoxyuridylate
(FdUMP)
•
•
•
•
Antitumor agent.
Irreversible inhibitor
of TS
Binds like dUMP but
in step 3 of the
reaction, F cannot
be extracted.
Suicide substrate.
F
FdUMP
Page 1091
Figure 28-20 The X-ray structure of the E. coli
thymidylate synthase–FdUMP–THF ternary complex.
Thymine synthase oxidizes N5,N10methyleneTHF
•
•
•
•
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 N5,N10methylene-THF and produces glycine.
Page 1091
Figure 28-21 Regeneration of N5,N10methylenetetrahydrofolate.
Nucleotide degradation
•
•
•
•
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 + H2O
Nucleoside + Pi
nucleosidase
Nucleoside
phosphorylase
base + ribose
base + ribose-1-P
Catabolism of purines
•
•
•
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).
Purine nucleoside + Pi
•
Purine nucleoside
phosphorylase
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
Page 1093
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
Page 1094
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.
Page 1094
Figure 28-24b
(b) The proposed
catalytic mechanism of adenosine deaminase.
1.
Zn2+ polarized H2O 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).
Purine nucleotide cycle
•
•
•
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.
Page 1095
Figure 28-25 The purine nucleotide cycle.
Xanthine oxidase
•
•
•
•
•
•
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 O2 which is converted to H2O2
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
Page 1095
Central
domain is
gold
C-terminal
domain is
lavender
Mechanism for XO
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.
Page 1096
Figure 28-27 Mechanism of xanthine oxidase.
Figure 28-23 Major pathways of purine catabolism in
animals.
ADA
Page 1093
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.
Page 1097
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.
Catabolism of pyrimidines
•
•
•
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).
Page 1098
Biosynthesis of of
NAD and NADP+
Page 1099
Produced from vitamin
precursors Nicotinate
and Nicotinamide and
from quinolinate, a Trp
degradation product
Biosynthesis
of FMN and
FAD from
riboflavin
Page 1100
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).
Page 1101
Biosynthesis of CoA
from pantothenate