Nucleotide Metabolism Flashcards
Nucleoside
Linking nitrogen base to 1’ position on ribose ring
Nucleotide
contains nucleoside esterfied to phosphate at 5’ position of ribose ring
deoxyribonucleotide
hydroxyl group at 2’ position of ribose ring
Pyrimidine vs Purine
Pyrimidine is ring w/ 2 nitrogens; Purine is ring w/ -N from pyrimidine but has 2 -N’s and 1 -C attached to form a side 5 member ring essentially
1st Step of Purine/Pyrimidine synthesis
generation of PRPP (5-phosphoribosyl-1-pyrophosphate); Ribose-5-Phosphate(from PPP Pathway)—-(PRPP synthetase)—>PRPP; PRPP is the base where the ring structure is assembled on; this steps commits ribose-5-P to nucleotide synthesis; NOTE it utilizes ATP!
Committed step of Purine synthesis
PRPP—(amidophosphoribosyltransferase)—> 5-phosphoribosylamine (PRA); uses gluatmine as nitrogen source
Pathway from PRA to inosine-5-phosphate (IMP): List requirements for it (4 of them)
9 steps (dont need to know them); but do know:
- carbon and nitrogen atoms are donated by AA’s (mainly glutamine, glycine and aspartate);
- CO2 provides both carbon and oxygen;
- N10-formyl THF serves as donor of one carbon groups;
- input of ATP energy is required at SEVERAL steps (6 ATPs counting formation of PRPP ribose-5-p from PPP pathway)
Synthesis of AMP: Regulation, Requirements
IMP—-(Adenylosuccinate Synthase + Aspartate + GTP)—>Adenylosuccinate—(Adenylosuccinase)—> AMP;
Adenylosuccinate synthase is rate limiting step and is inhibited by AMP; AMP synthesis uses GTP!
Synthesis of GMP: Regulation and Requirements
IMP—(IMP Dehydrogenase + NAD+)—> Xanithine-5-monophosphate—(GMP synthase+ glutamine + ATP)—> GMP;
IMP dehydrogenase inhibited by GMP; requires input of ATP!
nucleoside 5-monophosphate kinase
differing substrate specificities; NMP + ATP—> NDP + ADP
nucleoside 5-diphosphate kinase
broad substrate specificity; N1DP + N2TP—–> N1TP + N2DP
Points of Regulation in de novo synthesis of purines
1) GDP/ADP inhibit PRPP synthetase so PRPP is not produced from Ribose-5-Phosphate; supply of PRPP is reduced in response to an abundance of purine nucleotides; remember PRPP is needed for synthesis of BOTH purines and pyrimidines so GDP and ADP could also inhibit pyrimidine synthesis
2) AMP/GMP inhibit amidophosphoribosyltransferase; no formation of PRA from PRPP (committed step of purine synthesis); purine synthesis limited b/c….
3) PRPP is ALSO an allosteric activator of amidophosphoribosyltransferase so increase in PRPP stimulates de novo purine synthesis
GMP Degradation
- Phosphate removed by 5’ nucleotidases giving guanosine
- ribose removed by purine nucleoside phosphorylase; this generates free guanine base
- guanine deaminase removes -NH group producing Xanithine
- Xanithine oxidase generates uric acid
AMP Degradation (2 Paths)
1st Path:
1. AMP deaminase converts AMP—> IMP which is then degraded by 5’-nucleotidase and purine nucleoside phosphorylase (same as GMP)
2nd Path:
1. AMP converted to adenosine by 5’-nucleotidases
2. adenosine deaminase converts adenosine to inosine
3. inosine then converted to hypoxanthine by purine nucleoside phosphorylase
4. hypoxanthine converted to xanthine by xanthine oxdiase and again by XO into uric acid
Hyperuricemia
excess uric acid in blood; results from reduced excretion (most common) or increased production (less common); conditions that cause reduced excretion = renal insufficiency or metabolic acidosis; conditions that cause increased production = increased NT turnover (hemolytic diseases or chemotherapy) or diets rich in purines
Gout
uric acid = relatively insoluble; in normal conditions it is present in blood near saturation level; increase in levels cause crystals of sodium urate to deposit in joint; pain and swelling;
Kidney Stones and Uric Acid
high conc. of uric acid in urine can lead to deposition of uric acid stones (uric acid urolithiasis)
Allopurinol Treament
effective treatment for hyperuricemia; it is oxidized to oxypurinol which is competitive inhibitor of xanthine oxidase (build up of hypoxanthine(AMP) and xanthine(GMP)); both of these products are more soluble than uric acid and are excreted in kidneys
Pyrimidine vs. Purine Synthesis
unlike purines, pyrimidines are synthesized as free bases;
Pyrimidine Synthesis
Starts w/ synthesis of UMP:
1. Glutamine + CO2 + 2 ATP —–(Carbamoyl Phosphate Synthetase II)—>Carbamoyl Phosphate
2. —–(Aspartate + Aspartate Transcarbamoylase)—-> N-carbamoyl aspartate
3. —-(Dihydroorotase)—->Dihydroorotate
(FIRST 3 enzymes are part of CAD multifunctional protein)
4. —-(Dihydroorotate dehydrogenase[mito enzyme])—>Orotate
5. —-(Orotate Phosphoribosyltransferase + PRPP!!!!)—>OMP
6. —–(OMP decarboxylase)—-> UMP
(LAST 2 enzymes are also catalyzed by multifunctional protein known as UMP synthase)
Orotic Aciduria
defect in UMP synthase; orotic acid accumulates and is detected in urine; symptoms of megloblastic anemia and defects in cellular immunity due to inability to synthesize pyrimidines de novo; treatment = pyrimidine supplementation
Synthesis of CMP/CTP
UMP converted to UTP by nucleoside mono/diphosphate kinases;
UTP then converted to CTP by CTP synthase (requires addition of glutamine and ATP);
Regulation of de novo pyrimidine synthesis
PRPP upregulates carbamoyl phosphate synthetase II; UTP, however, downregulates CPSII;
UTP also UPregulates CTP synthase;
CTP inhibits CTP synthase
Synthesis of deoxyribonucleotides
must be synthesized in correct amounts;
catalyzed by ribonucletide reductase converting nucleoside-5-diphosphate—-> 2-deoxyribnucleotide 5-diphosphate;
Thioredoxin is oxidized in order to reduce the NDP–>dNDP;
Thioredoxin MUST be reduced again to continue makind dNDP’s by Thioredoxin Reductase w/ use of NADPH
Regulation of Ribonucleotide Reductase
complex regulation; transcriptional control modulates amount of enzymes in cell; dNTPs and NTPs can function as both an activator AND inhibitors (Ex. dATP inhibits reduction of ADP, GDP, CDP and UDP)’
Hydroxyuera Drug
inhibitor of ribonucleotide reductase; prevents dNTP synthesis and blocks cell division; important role for cancer chemotherapy
Severe Combined Immunodeficiency
group of disorders that impact both T- and B-cells; ex. is defect in adenosine deaminase (15%); leads to accumulation of dATP; this inhibits ribonucleotide reductase which blocks conversion of all purine and pyrimidine to 2-deoxynucleotides; effective immune response requires cell proliferation and w/o production of dNTP’s cell division CANNOT proceed
Synthesis of dTMP
no TMP, TDP or TTP in cells; only in deoxy form (dTMP, dTDP, dTTP); dTMP arises from dUMP;
catalyzed by thymidylate synthase (methylates dUMP using N5, N10 methylene THF);
dTDP/dTTP formed by nucleoside mono/diphosphate kinase
Inhibition of Thymidylate Synthase
5-Fluorouracil (analog of uracil) can be converted to FUMP and then to FUDP;
FUDP can be converted to FUTP which is incorporated into RNA (bad consquences for cells) or to FdUDP;
FdUDP can 1) be converted to FdUTP which can be incorporated into DNA and leads to strand breakage/cell death; or 2) be converted to FdUMP which can work w/ N5, N10-methylene THF and covalently bind to thymidylate synthase resulting in irreversible inhibition
Degradation of Pyrimidines: Metabolites and their uses
yields beta-AA’s; UMP, CMP, dCMP are converted to beta-alanine and dTMP degraded to beta-aminoisobutyrate; both are water soluble and can be excreted out or can be metabolized further w/ beta-alanine giving acetyl-CoA and beta-aminoisobutyrate giving succinyl-CoA;
Beta-Aminoisobutyrate Production
unique to thymine degradation; measurement of it in urine gives measure of DNA turnover; ex. increased levels found in urine of cancer patients undergoing chemo
Methotrexate Inhibitor
folate analog; inhibits dihydrofolate reductase; therefore there isnt production of N5, N10-methylene THF so thymidylate synthase is inhibited as well so no production of dTTP; lack of cell proliferation; cancer patients
De novo NT synthesis occurs where?
liver
Sources of free bases and nucleosides?
DIET and purine/pyrimidine NT degradation
Why is Glutamine important for intestinal epithelial cells?
b/c those cells are constantly dividing and glutamine is the -N group donor for purine/pyrimidine synthesis for DNA replication
Beginning of NT Salvage Pathway
- Ingest RNA/DNA
- pancreatic nucleases release free nucleoTIDES
- Phosphatases create free nucleoSIDES
- Taken up by epithelial cells and most are metabolized by the cells themselves since they are rapidly dividing
- Some free bases and nucleoSIDES released into circulation
- Traffic to tissues via RBC’s
Salvage of Purine Bases and NucleoSIDES: enzymes needed
require 2 enzymes: 1) hypoxanthine-guanine phosphoribosyltransferase (HGPRTase); 2)Adenine Phosphoribosyltransferase (APRTase); 1. Hypoxanthine + PRPP IMP + PPi 2. Guanine + PRPP GMP + PPi (1 and 2 rxns use HGPRTase) 3. Adenine + PRPP AMP + PPi (3 uses APRTase)
HGPRTase and APRTase are inhibited by what?
increases in IMP/GMP will causes compounds to compete w/ PRPP binding to HGPRTase;
same w/ AMP and APRTase
How do the salvage pathways decrease de novo synthesis?
- -PRPP is being consumed so amount of it available for generation of 5-phosphoribosylamine by amidophosphoribosyltransferase is significantly decreased (this is committed step of purine synthesis);
- –amidophosphoribosyltransferase rxn is inhibited by AMP/GMP
What is only purine nucleoside that can be directly phosphorylated to regenerate a NT?
adenosine by adensoine kinase
adenosine —-(ATP+Adenosine Kinase)—> AMP
Lesch-Nyhan Syndrome
characterized by hyperuricemia, uric acid urinary stones, intellectual disability and biting of fingers/lips;
deficiency of HGPRTase activity;
Salvage of Pyrimidine Bases: Rxn, Substrates, Regulation
Orotate, Uracil, or Thymine + PRPP Pyrimidine nucleoside 5-monophosphate (NMP);
catalyzed by pyrimidine phosphoribosyltransferase;
CYTOSINE IS NOT SALVAGED in humans!;
AGAIN, availability of PRPP regulates the de novo synthesis
Conversion of Pyrimidine Nucleosides to Nucleotides
occurs via specific kinases; ex. uridine-cytidine kinase
Uridine—-(ATP + enzyme)—>UMP
5-fluorocytosine
potent antifungal agent; fungi express cytosine deaminase which converts 5-fluorocytosine to 5-fluorouracil (exhibits same toxic effects as it does w/ thymidylate synthase, FUMP production and etc.); drug isnt harmful to humans
