Nucleotide Metabolism

Roles of nucleotides in the cell:
1) Activated precursors of DNA and RNA
2) Nucleotide derivatives are activated intermediates in many biosynthetic pathways
e.g. UDP-glucose, CDP-diacylglycerol
3) Universal currency of cell (i.e. ATP)
4) Components of three major coenzymes: NAD
+
, FAD, and CoA
5) Metabolic regulators (i.e. cyclic AMP)
Nucleotide synthesis can either by de novo or by recycling preformed bases (salvage pathway).
Nomenclature:
Nucleotides are composed of three components:
1) nitrogenous base - pyrimidine (cytosine, uracil, thymine) or purine (adenine or guanine)
2) pentose sugar - ribose (RNA) or deoxyribose (DNA)
3) phosphate group
nucleoside - purine or pyrimidine base linked to pentose sugar
nucleotide - phosphate ester of nucleoside
SYNTHESIS OF PURINE NUCLEOTIDES
Purine ring is synthesized de novo from 5 different precursors: aspartate (N-1 atom), CO2
(C-6 atom), glycine (C-4, C-5, N-7 atoms), tetrahydrofolate (C-2, C-8 atoms) and glutamine (
N-3, N-9).
Purine ring structure is synthesized from ribose 5-phosphate; PRPP then donates ribose 5-
phosphate for purine synthesis.
Purine ring is built onto the ribose 5-phosphate via a 10 -tep pathway: glutamine, glycine,
tetrahydrofolate, and glutamine make contributions to form 5-membered ring; construction
of 6-membered ring forms inosine 5’-monophosphate (IMP).
IMP can be converted into AMP or GMP
For AMP synthesis, aspartate amino group condenses with keto-group of IMP; GTPdependent
reaction
For GMP synthesis, C-2 is oxidized to form xanthosine monophosphate (XMP)
Amide nitrogen of glutamine replaces oxygen of C-2 to form GMP
ATP-dependent reaction
Synthesis of purine bases using the salvage pathway:
Free purine bases are formed by degradation of nucleic acids and nucleotides.
Purine nucleotides can be synthesized from preformed bases by salvage reactions (simpler
and less costly than de novo pathway).
2
Ribose phosphate portion of PRPP is transferred to purine to form the corresponding
ribonucleotide:
purine PPi
PRPP purine nucleotide
Two salvage enzymes recover purine bases:
1) adenine phosphoribosyl transferase
adenine + PRPP --------> adenylate + PPi
2) hypoxanthine-guanine phosphoribosyl transferase (HGPRTase)
hypoxanthine + PRPP --------> inosinate + PPi
guanine + PRPP ---------> guanylate + PPi
Regulation of purine nucleotide synthesis:
Probably largely by feedback inhibition.
Glutamine-PRPP amidotransferase (in main pathway) is allosterically inhbited by IMP, AMP, GMP.
Those steps leading specifically to AMP or GMP synthesis work primarily by feedback inhibition
XMP and GMP inhibit IMP dehydrogenase
AMP inhibits adenylosuccinate synthetase
SYNTHESIS OF PYRIMIDINE NUCLEOTIDES
Pyrimidine ring is assembled first, then linked to ribose phosphate ---> pyrimidine
nucleotide.
Requires fewer ATPs than purine synthesis ( 2 vs. 4).
Pyrimidine ring has three metabolic precursors: bicarbonate, amide group of glutamine,
aspartate.
PRPP is also required.
There is a 6-step pathway for de novo synthesis of UMP:
1) glutamine combines with bicarbonate ion + 2ATPs to yield carbamoyl phosphate +
glutamate
2) carbamoyl phosphate combines with aspartate via aspartate transcarbamolyase to form
carbamoyl aspartate (product contains all the atoms necessary for pyrimidine ring).
3) carbamoyl phosphate is cyclized enzymatically to form L- dihydroorotate.
4) L-dihydroorotate is oxidized by dihydroorotate dehydrogenase to form orotate; eremoved
from substrate are transferred to ubiquinone ---> O2 to ETS.
5) Orotate replaces pyrophosphate group of PRPP to form orotidine 5’-monophosphate(OMP)
3
6) OMP is decarboxylated by OMP decarboxylase to form uridine 5’-monophosphate (UMP)
Dihydroorotate is produced in the cytosol, then passes through the outer mitochondrial membrane.
Enzyme dihydroorotate DH is on outer surface of inner mitochondrial membrane
Orotate then moves back into cytosol
Regulation of UMP synthesis:
asparate carbamoylase (ATCase)- main regulatory enzyme
Inhibited by UTP and CTP
Activated by ATP
Keeps purines and pyrimidines in equal amounts
Synthesis of CTP
Formation of CTP from UMP in three reactions (see pathway sheet).
Regulation of pathway is via CTP synthetase
- allosterically inhibited by CTP
CONVERSION OF RIBONUCLEOTIDES TO DEOXYRIBONUCLEOTIDES
Deoxyribonucleotides are formed from ribonucleotides by ribonucleoside diphosphate reductase.
Energy to fuel reduction comes from NADPH.
There are really three proteins involved:
1) thioredoxin reductase
2) thioredoxin
3) ribonucleotide reductase
Once dADP, dGDP, and dCDP are formed, they are phosphorylated by nucleoside diphosphate
kinases.
Regulation of ribonucleoside diphosphate reductase is complex because there are 2 regulatory
sites:
1) Activity site - a.k.a. allosteric site - controls catalytic site
2) Specificity site - also allosterically regulated- controls substrate specificity
If ATP is bound in activity, enzyme is ACTIVE
If dATP or ATP is bound, reductase is pyrimidine specific
CDP --> dCDP
UDP --> dUDP
Binding of dTTP to specificity site causes enzyme to take GDP --> dGDP.
Binding of dGTP to specificity site causes enzyme to take ADP --> dADP.
4
Synthesis of Deoxythymidylate (dTMP) by Methylation of dUMP
dTMP is formed from dUMP, which is formed by any of the following:
dUDP + ADP dUMP + ATP enzyme is nucleoside monophosphate kinase
dUDP + ATP dUTP dUMP + PPi
dCMP + H2O dUMP + NH4
+
dUMP is converted to dTMP by thymidylate synthase
Methyl group donor is methylene tetrahydrofolate
Many cancer drugs inhibit the activity of thymidylate synthase and dihydrofolate
reductase --> decreased levels of dTMP synthesis --> decreased DNA synthesis
SALVAGE OF PURINES AND PYRIMIDINES
Purine Catabolism
Many organisms convert purine nucleotides to uric acid (see pathway sheet)
AMP ---> IMP ---> hypoxanthine ---->
GMP --> xanthine ----->
High serum levels of uric acid may lead to gout
Inflammation of joints is due to precipitation of sodium urate crystals
Kidneys may also be damaged by deposition of crystals
Gout is thought to be an inherited metabolic disease
Some patients with gout have a partial deficiency of HGPRTase
- leads to reduced synthesis of GMP and IMP by salvage pathway
- causes increase in PRPP levels --> increased purine biosynthesis by de novo pathway
Gout can also be caused by increased levels of PRPP caused by a yperactive synthetase
Gout can be treated with allopurinol, an analog of hypoxanthine --> ultimately acts as an
inhibitor of xanthine oxidase; called suicide inhibition
Lesch-Nyhan syndrome
Total lack of HGPRTase.
Results in compulsive self-destructive behavior.
Self-mutilation, mental deficiency, spasticity.
Elevated levels of PRPP ---> increased rate of purine biosynthesis by de novo pathway --->
overproduction of uric acid.
Possible that brain may rely heavily on salvage pathway for IMP and GMP synthesis.
5
Shows that abnormal behavior can be caused by absence of a single enzyme.
Pyrimidine Catabolism
Begins with the hydrolysis of nucleosides and Pi from nucleotides.
Successive reactions produce ribose 1-phosphate or deoxyribose 1-phosphate.
nucleotides
nucleosides Pi
ribose 1-phosphate
OR + thymine OR uracil
deoxyribose 1-phosphate
Thymine is ultimately broken down to succinyl CoA.
Uracil and cytosine are broken down into alanine, then acetyl CoA.