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FCH 532 Lecture 28
Chapter 28: Nucleotide metabolism
Quiz on Monday essential amino acids
Wed. April 11-Exam 3
ACS exam is on Monday 5/30
Final is scheduled for May 4, 12:45-2:45 PM, in
111 Marshall
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Figure 26-60 The
biosynthesis of the
“aspartate family” of
amino acids: lysine,
methionine, and
threonine.
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Figure 26-61 The
biosynthesis of the
“pyruvate family” of
amino acids:
isoleucine, leucine,
and valine.
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Figure 26-62 The
biosynthesis of
chorismate, the
aromatic amino acid
precursor.
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Figure 26-63
The
biosynthesis of
phenylalanine,
tryptophan, and
tyrosine from
chorismate.
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Figure 26-64 A ribbon diagram of the bifunctional
enzyme tryptophan synthase from S. typhimurium
Page 1045
Figure 26-65 The
biosynthesis of
histidine.
Hypoxanthine
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
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1.
7.
Chorsmate mutase
8.
Prephenate
dehydrogenase
9.
Aminotransferase
10.
Prephenate
dehydratase
11.
aminotransferase
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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 5phosphoribosylamine)
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.
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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.
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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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•
•
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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.