Nucleotide Metabolism
Robert J. Trumbly
Key to Objectives
1.
. Identify the structure of the five common nitrogenous bases found in nucleotides that are
incorporated into nucleic acids. Name the nucleosides and nucleotides derived from these
bases.
see figures.
2. Identify the nitrogenous base, sugar, phosphate, glycosidic bond, phophomonoester
bond, and phophoanhydride bonds of a nucleoside triphosphate.
see figures.
3. . Name the committed steps in the de novo purine and pyrimidine biosynthetic
pathways
Purine: PRPP glutamyl amidotransferase
Pyrimidine: carbamoyl phosphate synthetase II (CPSII)
4. Compare and contrast biosynthetic pathways for purines and pyrimidines.
both are made from simple compounds:
purines: CO2, aspartate, glycine, amide of glutamine, formyl THF
pyrimidines: CO2, aspartate, amide of glutamine
methylene THF required for TMP synthesis.
Both use PRPP for source of ribose.
purines: ring built on ribose phosphate.
pyrimidines: ribose 5-phosphate added to ring after it is built.
5. . Identify the principal metabolic source of one-carbon units that are metabolized via
tetrahydrofolate.
-carbon of serine, from serine hydroxy methyl transferase reaction. (page 41)
6. State the first purine nucleotide that is formed during de novo purine biosynthesis
first purine nucleotide made: IMP, inosine monophosphate
7. State the first pyrimidine nucleotide that is formed during de novo pyrimidine
biosynthesis
first pyrimidine nucleotide made: OMP, orotidine monophosphate
8. Name the two steps that are regulated early in the de novo purine biosynthetic pathway.
PRPP synthetase and PRPP glutamyl amidotransferase.
9. . Name the enzyme that is regulated early in the de novo pyrimidine pathway of
mammals.
: carbamyl phosphate synthetase II.
10. . State the enzyme of nucleotide metabolism that is inhibited by a given
antimetabolite..
a. glutamine analogs inhibit amidotransferases in purine biosynthesis:
PRPP glutamyl amidotransferase = amidophosphoribosyl transferase
phosphoribosyl-formyl glycine amidine synthetase.
Glutamine analogs also inhibit CTP synthetase using glutamine as amino donor in the
pyrimidine synthetic pathway.
Therefore glutamine analogs inhibit both purine and pyrimidine biosynthesis.
b. purine nucleotide analogs such as mercaptopurine are converted to ribonucleotides,
inhibit PRPP glutamyl amidotransferase.
c. sulfa drugs inhibit folate synthesis in bacteria, by substituting for p-aminobenzoic acid
in the reaction catalyzed by dihydropteroate synthase.
d. Antifolates including methotrexate and aminopterin inhibit dihydrofolate reductase
(DHFR), resulting in a depletion of tetrahydrofolate.
e. 5-fluorouracil (5-FU): thymidylate synthase
11. State the level of phosphorylation at which ribonucleotides are converted to
deoxyribonucleotides.
diphosphate.
12. State which nucleotides allosterically inhibit ribonucleotide reductase, and why high
levels of thymidine arrests growth of cells.
Regulation of ribonucleotide reductase is very complicated.
points to know:
a. There are two allosteric sites: an overall activity site, and a substrate specificity site.
dATP inhibits at the general activity site.
b. dGTP and dTTP also act as inhibitors at the specificity site.
dTTP inhibits reduction of pyrimidine nucleoside diphosphates.
c. why does thymidine arrest growth of cells?
Answer: thymidine added to tissue culture cells at a conc. greater than 1mM blocks DNA
synthesis. The pools of dTTP, dGTP, and dATP are greatly increased, the pool of dCTP
decreased 10-fold. The primary reason is that dTTP inhibits the reduction of pyrimidine
nucleoside diphosphates. dTTP can be made from thymidine
13. . Describe the thymidylate synthase reaction and its role in nucleotide synthesis
Thymidylate synthase reaction: dUMP + N5-N10-methylene THF -> TMP + DHF.
Only pathway for TMP synthesis. Oxidizes folate which must be returned to the reduced
state by DFHR.
14. . Diagram the two “preformed” or “salvage” pathways of nucleotide biosynthesis:
a. phosphoribosyl transferases
PPRP + adenine -> (APRT) AMP + PPi
PPRP + guanine -> (HGPRT) GMP + PPi
PPRP + hypoxanthine -> (HGPRT) IMP + PPi
b. nucleoside phosphorylase and kinases
thymine + deoxyribose-1-P <-> (NP) thymidine + Pi
thymidine + ATP -> (NK) dTMP + ADP
doesn't work for cytosine, even though there is a deoxycytidylate kinase, since there is no
phosphorylase for cytidine. In the pyrimidine catabolic pathway, cytidine is converted to
uridine, which is then subjected to phosphorylase, so no cytidine phosphorylase.
15. State the end product of purine catabolism in man and the precursors that xanthine
oxidase acts upon to yield this end product.
end product of purine catabolism: uric acid.
hypoxanthine->xanthine->uric acid
16. State the end products of pyrimidine catabolism in man.:
CO2, NH3, -alanine, and -aminoisobutyrate.
17. State the known enzymatic defects in patients with Gout, Lesch-Nyan Syndrome,
Orotic Aciduria, Xanthinuria, and Severe Combined Immunodeficiency:
Gout
PPRP synthetase, deregulated.
HGPRTase
glucose-6-phosphatase.
Lesch-Nyhan: HGPRTase.
Orotic aciduria: OMP-pyrophosphorylase, OMP-decarboxylase.
Xanthinuria: xanthine oxidase.
SCID: adenosine deaminase
other immunodeficiency: purine nucleoside phosphorylase
Treatments:
Gout
Allopurinol, uricosurics (probenecid), anti-inflammatory agents (colchicine, NSAIDs)
Lesch-Nyan
HGPRTase deficiency.
allopurinol will relieve hyperuricemia, not neurological symptoms.
Orotic aciduria. defect in UMP synthase.
effective treatment: oral uridine.
Xanthinuria defect in xanthine oxidase
treatment: low purine diet and high fluid uptake.
SCID adenosine deaminase deficiency
treatment: ADA-PEG conjugates, gene therapy with ADA gene, bone marrow transplant.