BIOCHEMISTRY NOTES

Purine and Pyrimidine Nucleotide Metabolism (Devlin)

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Introduction
 Cellular levels of purine and pyrimidine
nucleotides are maintained by de novo synthetic
pathways and salvage reactions
 Sources for C, O, N
o Amino acids
o “carbon-1”-tetrahydrofolate
o Ribose-5-P
 Intracellular concentrations of nucleotides are
finely controlled by allosterically regulated
enzymes in pathways
 Nucleotide end products of pathways serve as
effectors and regulate key steps in pathways
 2’-deoxyribonucleotides for DNA replication
o Generated directly from ribonucleotides
o Production is carefully regulated by
nucleoside 5’-triphosphate nucleotides
acting as (+) and (-) effectors
 Concentrations of key enzymes in nucleotide
metabolic pathways are altered during the cell
cycle due to increases in enzyme activity especially
during late G1/early S phase prior to DNA
replication
Metabolic functions of nucleotides
 Hypoxic cells – nucleoside 5’-monophosphates
and nucleoside 5’-diphosphates are greatly
increased
 Endogenous or exogenous nucleotide/nucleic acid
degradation products
o Free nucleobases
o Nucleosides
o Nucleoside 2’- and 3’-monophosphates
o Modified bases
 [ribonucleotides] >>>> [2’-deoxyribonucleotides]
o HOWEVER, during DNA replication:
↑[2’-deoxyribonucleotides]
 The total concentration of nucleotides in normal
cells is essentially constant
o [AMP + ADP + ATP] is constant
o But it doesn’t mean the ATP/(ATP +
ADP+ AMP) ratio is constant
 Depends on cell’s energy state
o Rule applies for NAD+ and NADH
o De novo synthesis and salvage pathway
maintain fixed concentration of
nucleotides
5’-phosphoribosyl-1-pyrophosphate and glutamine in
de novo synthesis of nucleotides
5’-phosphoribosyl-1-pyrophosphate (PRPP)

 Ribose-5-phosphate
o Generated from PPP (from glucose-6-P)
o Supplies PRPP for de novo and salvage
pathways
o Competitively inhibited by 2,3-
bisphosphoglycerate
 Ribose-5-phosphate  PRPP
o ENZYME: PRPP synthetase
 Inhibited by bis-2,3-DPG
o Requires: ATP, Mg2+
 ADP is a competitive inhibitor to
Distribution of nucleotides varies with cell types ATP
o Rxn is tightly regulated
 Principal purine and pyrimidine compounds found  Involved in:
in cells are the 5’-nucleotide derivatives o De novo synthesis of purine and
o ATP has the highest concentration pyrimidine nucleotides
 RBC – adenine nucleotides o Salvage of purine and pyrimidine bases
 Liver – complete spectrum of nucleotides o NAD synthesis
 Normally fxning cells – nucleoside 5’-
triphosphates predominate

Glutamine
 Critical substrate in five specific rxns involved in
the de novo synthesis of nucleotides
 The de novo synthesis of purine and pyrimidine
nucleotides are highly regulated at PRPP
amidotransferase and CPS II
 Regulation of CTP synthetase = important for
maintaining cellular ratio of UTP to CTP
Synthesis of purine nucleotides
 All enzymes of the purine nucleotide pathway are
cytosolic
o For pyrimidines, both cytosolic and
mitochondrial
 Energy from hydrolyzing ATP drives several
reactions
o Making purines and pyrimidines is
EXPENSIVE, lots of ATP needed
 Not all cells (RBCs) are capable of de novo purine
synthesis
De novo pathway for purine synthesis
 Ultimately synthesizes IMP
 IMP – common precursor for AMP and GMP
 Pathway is highly regulated by AMP, GMP, IMP
 Requires amino acids (serine, glycine, tryptophan,
histidine) as carbon and nitrogen donors, CO2 as a
carbon source, and C1-units transferred via H4-
folate
Formation of IMP
 6 mol ATP utilized per mol IMP synthesized
(EXPENSIVE)
 Formation of 5-phosphoribosylamine – first step,
committed, and regulated step
o N-C bond formed will be the N-glycosidic
bond of the purine nucleotide
 Two reactions in the pathway (3 and 9) use
tetrahydrofolate as a carbon carrier which gets
regenerated
 Phosphoribosyl-5-aminoimidazole carboxylase is
not a biotin-dependent carboxylase
Formation of AMP or GMP
 Conversion of IMP is regulated to maintain
appropriate cellular ratios of adenine and guanine
nucleotides
 IMP  GMP
o ATP needed as energy source
o Enzyme: IMP dehydrogenase (IMPDH)
 Rate-limiting
 IMPDH-I is constitutive; -II is
related to cell growth and
proliferation
 IMP  AMP
o GTP needed as energy source
o Enzyme: Adenylsuccinate synthetase
 Rate-limiting
 AMP is a competitive inhibitor of
IMP
AMP or GMP  ATP or GTP
 Enzyme: nucleoside 5’-monophosphate kinases
and nucleoside 5’-diphosphate kinases
o Not rate limiting
Regulation of purine nucleotide synthesis
 RECALL: 5-phosphoribosylamine formation is the
committed step in purine nucleotide synthesis
 Glutamine PRPP-amindotransferase is rate
limiting
o Allosterically regulated by IMP, GMP, and
AMP (negative effectors)
 Forms a dimer (inactive form)
o PRPP is a positive effector of the enzyme
  Favors active monomeric form of
the enzyme
o Has distinct nucleotide binding sites
(oxypurine nucleotides and aminopurine
nucleotides have different binding sites
from each other)
 Methotrexate – drug used exclusively in cancer
treatment
o Cytotoxic – alters folate pools
 [adenine nucleotides] x4 of [guanine nucleotides]
 Gout – overproduction of purine nucleotides 
overproduction of uric acid
Salvage of purine bases and nucleosides to form
nucleotides
 Two salvage pathways of nucleobases
o Hypoxanthine-guanine phosphoribosyl
transferase (HGPRTase)
 IMP and GMP are competitive
inhibitors to PRPP
 Enzyme is depressed in the Lesch-
Nyhan syndrome
 Hyperuricemia
 Mental retardation
 Self-mutilation
o Adenine phosphoribosyl transferase
(APRTase)
 AMP is a competitive inhibitor to
PRPP
 Deficiency causes increased
excretion of adenine, 8-
hydroxiadenine, and 2,8-
dehydroxiadenime
 8-HA and 2,8-DHA are
generated by xanthine
oxidoreductase
 A,8-DHA accumulation
may form calculi
o Pathways regulated by end-products
 Hypoxanthine and guanine for salvage arise from
degradation of endogenous or exogenous purine
nucleotides
 Adenine in the salvage pathway is generated
mainly from the synthesis of polyamines
 Generating AMP and GMP through salvage
pathways decrease the de novo pathway activity
o PRPP gets consumed in salvage pathways,
decreasing the rate of formation of 5-
phosphoribosylamine
o AMP, GMP, IMP are negative effectors of
PRPP amidotransferase
 Nucleoside salvaging
o Adenosine
 Enzyme: adenosine kinase
 5’-phosphotransferase
 ATP as phosphate donor
 RBCs depend on purine phosphoribosyl
transferases and 5’-phosphotransferase to replenish
nucleotide pools
Interconversion of purine nucleotides
 No direct one-step pathway to convert GMP to
AMP and vice versa
 AMP or GMP  converted back to IMP
o Reductive deamination of GMP by GMP
reductase
 GMP activates this step;
xanthosine 5’-monophosphate
(XMP) strongly inhibits rxn
 GMP lowers Km and increases
Vmax
o AMP deaminase (5’-AMP aminhydrolase)
 Activated by K+ and ATP
 Inhibited b Pi, GDP, GTP
Formation of Uric Acid
 The degradation of purine nucleotides, nucleosides,
and nucleobases follow a common pathway that
leads to the formation of uric acid
 o Nucleases are specific toward either RNA
or DNA and also toward the bases and
position of cleavage site at the 3’,5’-
phosphodiester bonds
o Nucleotidases range from high specificity
(5’-AMP nucleotidase) to broad specificity
(acid and alkaline phosphatases)
o AMP deaminase – specific for AMP
o Adenosine deaminase – less specific
 Purine nucleoside phosphorylase catalyzes the ff.
reversible rxns:
 Removal of deoxyguanosine prevents uncontrolled
accumulation of dGTP, which is toxic to cells at
high concentrations
 Hypoxanthine and xanthine are oxidized by
xanthine oxidoreductase (both a DH and oxidase)
o DH requires NAD as the electron acceptor;
oxidase utilizes molecular oxygen and
generates H2O2 as a product
 Xanthine oxidoreductase contains FAD, Fe, and
Mo
 Uric acid is the unique end product of purine
nucleotide degradation
o Not very soluble in aqueous medium
Metabolism of pyrimidine nucleotides
 De novo synthesis of the pyrimidine ring utilizes
amino acids as carbon and nitrogen donors in
addition to CO2
 UMP is synthesized in the metabolic pathway
 Not all enzymes for de novo synthesis are cytosolic
o 5 out of 6 rxns take place in the cytosol of
the cell
 Pyrimidine ring is formed first and then ribose-5-P
is added with PRPP as the donor
 Carbamoyl phosphate synthetase I (CPS I) is found
in the mitochondria – functions as a part of the urea
cycle
 Formation of N-carbamoyl aspartate –
COMMITTED STEP
 Formation of CPS II – REGULATED STEP
 Formation of orotate from dihydroorotate
o Enzyme: mitochondrial Dihydroorotate
dehydrogenase (DHODH) – outer surface
of inner mitochondrial membrane
 Pyrimidine nucleotide synthesis is inhibited under
conditions in w/c mitochondrial respiration is
reduced
 CPS II, aspartate carbamoyl transferase,
dihydroorotase – found on a trifunctional protein
CAD
 Orotate phosphoribosyltransferase, OMP
decarboxylase – found in bifunctional protein UMP
synthase
 Nucleotide kinases convert UMP to UTP
 UTP – direct substrate for CTP synthetase
o Glutamine – amino group donor
Regulation of Carbamoyl Phosphate Synthetase II
 Cytosolic enzyme (distinct from CPS I)
 Inhibited by UTP, activated by PRPP
 Only source of carbamoyl phosphate in
extrahepatic tissues
 UMP does not inhibit carbamoyl phosphate
synthetase II; competes with OMP to inhibit OMP
decarboxylase
Salvaging of pyrimidine bases to reform nucleotides
 Enzyme: pyrimidine phosphoribosyltransferase
 Utilizes orotate, uracil, thymine, NOT CYTOSINE
Formation of Deoxyribonucleotides

 By reduction of ribonucleoside 5’-diphosphates

 Enzyme: nucleoside 5’-diphosphate reductase
o Converts ribonucleoside  2’-
deoxyribonucleoside
o Consists of 2 non-identical subunits
 R1 – 2 different effector binding
sites
 R2 – nonheme iron and stsable
tyrosyl free radical
 Encoded by genes on separate
chromosomes
