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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