Purine and Pyrimidine nucleotide Metabolism Chutima Talabnin Ph.D. School of Biochemistry ,Institute of Science, Suranaree University of Technology 1
Purine and Pyrimidine nucleotide Metabolism
Chutima Talabnin Ph.D. School of Biochemistry ,Institute of Science, Suranaree University of Technology 1
Metabolic functions of nucleotide
• Main function act as a precorsors of DNA and RNA • Nucleotide and their derivatives play critical and diverse roles in cellular metabolism
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Digestion
DNA + RNA + Protein
DNA+RNA
Acidic condition in stomach
Protein
Nuclease from pancrease (deoxyribonuclease and ribonuclease)
Oligonucleotide
nucleoside monophosphate, NMP
Phosphodiesterase and Polynucleotidase from pancrease and intestine
nucleoside Phosphate group
Nucleotidase from intestine
Pentose sugar Nitrogenous base
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Purine or Pyrimidine nucleotide synthesis
De novo pathway : utilizes metabolic precursor including amino acids, ribose 5’ phosphate, NH3, CO2
etc. which consider expensive in term of mole of ATP utilize per synthesis
Salvage pathway : recycle the free bases or nucleoside both purine and pyrimidine from nucleic acid breakdown to reform nucleotide again
Purine or Pyrimidine ribonucleotide
Purine or Pyrimidine deoxyribonucleotide
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Purine ribonucleotide synthesis by De novo pathway
Begining with PRPP synthesis
De novo pathway for purine ring in mammalian cells utilize • Amino acid as a carbon and nitrogen donor • Tetrahydrofolate and CO2 as a carbon donor
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De novo pathway
Committed step or rate limiting step Enzyme is controlled by their products (feedback inhibition) including IMP, AMP และ GMP
Rate limiting Enzyme PRPP glutamyl aminotransferase (Glutamine PRPP aminotransferase act by moving amide group (NH2) from Glutamine to replace pyrophosphose group of PRPP
IMP (Inosine monophosphate) is the first purine ribonucleotide
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AMP and GMP synthesis
De novo pathway
IMP --- > Adenosine monophosphate (AMP) • Adding –NH2 from aspartate at position C6 of IMP • Adenylosuccinate synthetase • GTP
IMP --- > Guanosine monophosphate (GMP) • IMP --- > XMP (Xanthosine monophosphate) • Adding –NH2 from Glutamine at position C2 of IMP • Glutamine amidotransferase • ATP
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Regulation : Purine synthesi by De novo pathway
IMP (Inosine monophosphate) AMP (Adenosine monophosphate) GMP (Guanosine monophosphate)
De novo pathway
GDP
ATP GTP
ADP GDP
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Pyrimidine ribonucleotide synthesis by De novo pathway
Beginning with Carbamoyl phosphate (CAP) • CO2 (HCO3-) + Glutamine + ATP • Carbamoyl phosphate synthetase II (CSPII) in cytoplasm
De novo pathway for purine ring in mammalian cells utilize • Amino acid as a carbon and nitrogen donor • CO2 as a carbon donor
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Committed step or rate limiting step in E coli Enzyme is controlled by their products (feedback inhibition) including CTP
Rate limiting Enzyme in E.coli Aspartate transcarbamoylase
Adding PRPP to connect with Orotic acid (Orotate)
It lead to produce the first pyrimidine ribonucleotide which is OMP (orotidine monophosphate = orotidylate)
UMP and CTP synthesis
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UDP UTP Carbamoyl phosphate synthetase II (CSPII)
CTP Aspartate transcarbamoylase
Regulation : Pyrimidine by De novo pathway
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Digestion
DNA + RNA + Protein
DNA+RNA
Acidic condition in stomach
Protein
Nuclease from pancrease (deoxyribonuclease and ribonuclease)
Oligonucleotide
nucleoside monophosphate, NMP
Phosphodiesterase and Polynucleotidase from pancrease and intestine
nucleoside Phosphate group
Nucleotidase from intestine
Pentose sugar Nitrogenous base
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Purine and Pyrimidine synthesis by Salvage pathway
Base + PRPP Nucleoside-5'-monophosphate (NMP) + Pi (Purine or Pyrimidine base)
• Adenine phosphoribosyltransferase (APRT)
• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Adenine + PRPP -----> AMP + PPi
Guanine + PRPP -----> GMP + PPi
Hypoxanthine + PRPP -----> IMP + PPi
Nucleoside + ATP Nucleoside-5'-monophosphate (NMP) + NDP
Nucleoside kinase
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Diphosphate and Triphosphate synthesis
Adenylate kinase: AMP + ATP ---> 2 ADP Guanylate kinase: GMP + ATP ---> GDP + ADP
GDP + ATP ---> GTP + ADP
Nucleotide Monophosphate (NMP)
Nucleotide Diphosphate (NDP)
Nucleotide Triphosphate (NTP)
Nucleoside monophosphate kinase Nucleoside diphosphate kinase
ATP ADP ATP ADP
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Deoxyribonucleotide formation
• Deoxyribonucleotides are the building block of DNA and are derived from corresposding ribonucleotides by direct reduction at 2’ carbon atom of D-ribose to form 2’ D-deoxyribose derivative
• Ribonucleotide Reductase (Nucleoside 5’
diphophate reductase) catalyzed the reaction in which ribonucleotide Diphosphate (NDP) including ADP, GDP, CDP, and UDP are converted to 2’ deoxyribonucleotide 5’ diphosphate
• The reduction reaction of ribonucleotide reductase require a pair of hydrogen atoms which are ultimately donated by NADPH via intermediate hydrogen carrying protein, Thioredoxin or Glutaredoxin.
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Ribonucleotide Reductase • Dimer including R1 and R2 subunit
• Active thiol group on R1 react with 2’NAD to produce 2’deoxyribonucleotide and water
• Active site radical (-Xº ) on R2 help to stabilize NAD at 3’ ribonucleotide
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Regulation : Ribonucleotide Reductase (allosteric enzyme)
Ribonucleotide Reductase
dATP
ATP dGTP dTTP
CDP ---> dCDP UDP ---> dUDP
ADP ---> dADP GDP ---> dGDP
Primary regulation Activator = ATP Inhibitor = dATP
Substrate specific site ATP, dATP, dGTP, dTTP
Regulation of E.coli ribonucleotide reductase has two levels including enzyme activity and substrate specificity. This regulation is designed to provide a balance pool of precursors for DNA synthesis
dATP
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UDP + NADPH + H+ dUDP +NADP+
Ribonucleotide reductase
dUDP dUMP + Pi
Tetrahydrofolate (FH4) Dihydrofolate (FH2)
Thymidylate synthetase
NADPH + H+ NADP+
Dihydrofolate reductase
Methyl group (CH3) from tetrahydrofolate (FH 4)
dTMP (TMP) synthesis
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Chemotherapeutic drugs target enzyme in Nucleotide biosynthesis
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Purine degradation
Primates, Brids, Repetiles, Insects
Most mammals Urate oxidase
Bony fishes, Amphibians Allantoicase
Marine, Invertibate Urease (Ammonia)
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Lesch-Nyhan Syndrome: HGPRT Deficiency
Gout: An Excess of Uric Acid
Guanine + PRPP -----> GMP + PPi
Hypoxanthine + PRPP -----> IMP + PPi
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