FCH 532 Lecture 29
Chapter 28: Nucleotide metabolism
Chapter 24: Photosynthesis
New study guide posted
Forms of pyridoxal-5¢phosphate.
(c) Pyridoxamine-5¢-phosphate (PMP) and (d) The Schiff
base that forms between PLP and an enzyme -amino
group.
Page 986
Figure 26-1cd
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Page 997
Figure 26-13 The serine dehydratase reaction.
1. Formation of Ser-PLP Schiff base, 2. Removal of the -H atom of serine, 3.  elimination
of OH-, 4. Hydrolysis of Schiff base, 5. Nonenzymatic tautomerization to the imine, 6.
Nonenzymatic hydrolysis to form pyruvate and ammonia.
Serine hydroxymethyltransferase
catalyzes PLP-dependent C-C
cleavage
•
•
Catalyzes the conversion
of Thr to Gly and
acetaldehyde
Cleaves C-C bond by
delocalizing electrons of
the resulting carbanion into
the conjugated PLP ring:
H
B:
O H
H3C-HC--C-COON
H
C
2-O
H
O-
3PO
+
N
H
CH3
Page 1033
Figure 26-54 The
syntheses of alanine,
aspartate, glutamate,
asparagine, and
glutamine.
Page 1037
Figure 26-58 The conversion of
glycolytic intermediate 3phosphoglycerate to serine.
1. Conversion of 3phosphoglycerate’s 2-OH
group to a ketone
2. Transamination of 3phosphohydroxypyruvate
to 3-phosphoserine
3. Hydrolysis of
phosphoserine to make
Ser.
Purine synthesis
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•
Purine components are derived from various sources.
First step to making purines is the synthesis of inosine
monophosphate.
De novo biosynthesis of purines: low molecular weight
precursors of the purine ring atoms
Initial derivative is Inosine
monophosphate (IMP)
•
AMP and GMP are synthesized from IMP
H
O
-O
P
O-
Inosine monophosphate
Hypoxanthine
base
Inosine monophosphate (IMP) synthesis
•
•
•
•
•
Pathway has 11 reactions.
Enzyme 1: ribose phosphate pyrophosphokinase
Activates ribose-5-phosphate (R5P; product of pentose phosphate
pathway) to 5-phosphoriobysl--pyrophosphate (PRPP)
PRPP is a precursor for Trp, His, and pyrimidines
Ribose phosphate pyrophosphokinase regualtion: activated by PPi and
2,3-bisphosphoglycerate, inhibited by ADP and GDP.
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1. Activation of ribose-5phosphate to PRPP
2. N9 of purine added
Anthranilate
synthase
2.
Anthranilate
phosphoribosyltrans
ferase
3.
N-(5’phosphoribosyl)
anthranilate
isomerase
4.
Indole-3-glycerol
phosphate synthase
5.
Tryptophan
synthase
6.
Tryptohan synthase,
 subunit
Page 1043
1.
7.
Chorsmate mutase
8.
Prephenate
dehydrogenase
9.
Aminotransferase
10.
Prephenate
dehydratase
11.
aminotransferase
Page 1045
1.
ATP
phosphoribosyltransferase
2.
Pyrophosphohydrolase
3.
Phosphoribosyl-AMP
cyclohydrolase
4.
Phosphoribosylformimino-5aminoimidazole carboxamide
ribonucleotide isomerase
5.
Imidazole glycerol phosphate
synthase
6.
Imidazole glycerol phosphate
dehydratase
7.
L-histidinol phosphate
aminotransferase
8.
Histidinol phosphate
phosphatase
9.
Histidinol dehydrogenase
Page 1074
Nucleoside diphosphates are synthesized
by phosphorylation of nucleoside
monophosphates
Nucleoside diphosphates
•
Reactions catalyzed by nucleoside monophosphate kinases
Adenylate kinase
AMP + ATP
2ADP
Guanine specific kinase
GMP + ATP
•
GDP + ADP
Nucleoside monophosphate kinases do not discriminate
between ribose and deoxyribose in the substrate (dATP or
ATP, for example)
Nucleoside triphosphates are synthesized by phosphorylation
of nucleoside monophosphates
Nucleoside diphosphates
•
Reactions catalyzed by nucleoside diphosphate kinases
Adenylate kinase
ATP + GDP
•
ADP + GTP
Can use any NTP or dNTP or NDP or dNDP
Regulation of purine biosynthesis
•
•
•
•
•
•
Pathways synthesizing IMP, ATP and GTP are individually regulated in most
cells.
Control total purines and also relative amounts of ATP and GTP.
IMP pathway regulated at 1st 2 reactions (PRPP and 5-phosphoribosylamine)
Ribose phosphate pyrophosphokinse- is inhibited by ADP and GDP
Amidophosphoribosyltransferase (1st committed step in the formation of
IMP; reaction 2) is subject to feedback inhibition (ATP, ADP, AMP at one site
and GTP, GDP, GMP at the other).
Amidophosphoribosyltransferase is allosterically activated by PRPP.
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1. Activation of ribose-5phosphate to PRPP
2. N9 of purine added
Figure 28-5
Control
network for the
purine biosynthesis
pathway.
Page 1075
Feedback
inhibition is
indicated by
red arrows
Feedforward
activation by
green arrows.
Salvage of purines
•
•
•
•
Free purines (adenine, guanine, and hypoxanthine) can be reconverted
to their corresponding nucleotides through salvage pathways.
In mammals purines are salvaged by 2 enzymes
Adeninephosphoribosyltransferase (APRT)
Adenine + PRPP  AMP + PPi
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Hypoxanthine + PRPP  IMP + PPi
Guanine + PRPP  GMP + PPi
Synthesis of pyrimidines
•
•
•
•
Pyrimidines are simpler to synthesize than purines.
N1, C4, C5, C6 are from Asp.
C2 from bicarbonate
N3 from Gln
•
Synthesis of uracil monoposphate (UMP) is the first step for
producing pyrimidines.
Page 1077
Figure 28-6 The
biosynthetic origins of
pyrimidine ring atoms.
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Reaction 4: Oxidation of dihydroorate
Reactions catalyzed by eukaryotic dihydroorotate
dehydrogenase.
Oxidation of dihydroorotate
•
•
•
Irreversible oxidation of dihydroorotate to orotate by dihydroroorotate
dehydrogenase (DHODH) in eukaryotes.
In eukaryotes-FMN co-factor, located on inner mitochondrial membrane.
Other enzymes for pyrimidine synthesis in cytosol.
Bacterial dihydroorotate dehydrogenases use NAD linked flavoproteins
(FMN, FAD, [2Fe-2S] clusters) and perform the reverse reaction
(orotate to dihydroorotate)
Page 1079
Figure 28-9 Reaction 6: Proposed catalytic
mechanism for OMP decarboxylase.
Decarboxylation to form UMP involves OMP
decarboxylase (ODCase) to form UMP.
Enhances kcat/KM of decarboxylation by 2 X 1023
No cofactors
Synthesis of UTP and CTP
•
•
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
Page 1080
Figure 28-10 Synthesis of
CTP from UTP.
CTP is formed by amination of UTP by CTP
synthetase
In animals, amino group from Gln
In bacteria, amino group from ammonia
Regulation of pyrimidine
nucleotide synthesis
•
•
•
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.
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Page 1080
Production of deoxyribose derivatives
•
•
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).
Page 1082
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.
Thioredoxin and glutaredoxin
•
•
•
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•
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)
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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
Page 1091
Figure 28-20 The X-ray structure of the E. coli
thymidylate synthase–FdUMP–THF ternary complex.
Thymine synthase oxidizes N5,N10methyleneTHF
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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.