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Home , Thymidine monophosphate, Xanthosine monophosphate

Author(s): Dr. Robert Lyons, 2009

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Dr. Robert Lyons Assistant Professor, Biological Chemistry Director, DNA Sequencing Core

Web: http://seqcore.brcf.med.umich.edu/mcb500

Fall 2008 Amino Acid metabolism metabolism Amino acids

Met Methylene Cycle Glu, Gln, THF Asp, NH3

Urea

oxaloacetate

DNA P u rin e s P y rim id in e s RNA

Uric Acid (energy) fumarate TCA Cycle metabolism

R. Lyons Click on any blue rectangle to see details.

am i n o a c id s , Carbamoyl fo la t e PRPP

Purine Salvage IM P O M P Biosynthesis

Pyrimidine Salvage

P u r in e M P P y r im id in e M P

Ribonucleotide reductase reductase dNTP DNA dNTP NTP RNA NTP

Purine Pyrimidine Degradation Degradation

NH4 U ric A cid ( e n e rg y )

R. Lyons Formation of PRPP: Phosphoribose pyrophosphate

O P O O P O OH O P O P OH OH OH OH ATP AMP ribose - 5 - phosphate phosphoribose - 1 pyrophosphate R. Lyons

PRPP Use in Purine Biosynthesis:

H2O O O P O P O NH2 O P O P OH OH glutamine glutamate OH OH phosphoribose - 1 pyrophosphate R. Lyons O N HN

The First Purine: Monophosphate N N ( are involved in this synthesis) O P O

OH OH Conversion to : R. Lyons

O NH2 GTP GDP + Pi N N HN N

N N N N aspartate fumarate ribose-5-P ribose-5-P Inosine monophosphate R. Lyons Conversion to :

H O + 2 O NAD + O O NADH + H ATP AMP + PPi N N N HN N HN

N N O N N NH N N H 2 gln glu ribose-5-P ribose-5-P ribose-5-P Inosine monophosphate monophosphate R. Lyons Monophosphate

AMP + ATP <--> 2ADP (adenylate )

GMP + ATP <---> GDP + ADP (guanylate kinase)

• similar enzymes specific for each nucleotide • no specificity for ribonucleotide vs. Ribonucleotide Reductase

X O P O P O Enzyme• NADH

OH OH

X O P O P O Enzyme NAD+

OH H

R. Lyons

Hydroxyurea inhibits this enzyme: chemotherapeutic use

O

HONH C NH 2 Regulation of Ribonucleotide Reductase

ATP + CDP (-) dCDP dCTP

ATP + UDP (-) dUDP dTTP

GDP + dGDP dGTP

ADP + dADP dATP (-)

R. Lyons Nucleoside Diphosphate Kinase

N1DP + N2TP <--> N1TP + N2DP

dN1DP + N2TP <--> dN1TP + N2DP

• No specificity for base • No specificity for ribo vs deoxy Feed-forward regulation by PRPP

PRPP

IMP

ATP GTP

GMP AMP

GTP ATP

R. Lyons Feed-forward regulation by PRPP

PRPP +

IMP

ATP GTP GMP AMP

GTP ATP

R. Lyons Feed-forward regulation by PRPP

PRPP

IMP

ATP GTP + + GMP AMP

GTP ATP

R. Lyons Feed-forward regulation by PRPP

PRPP + - -

IMP - -

ATP GTP GMP AMP

GTP ATP

R. Lyons Degradation of the Purine :

+ NH H O NH O ribose - 1 - P O 2 2 4 Pi N N N N HN HN Adenosine purine nucleoside N N N N N N deaminase phosphorylase H ribose (ADA) ribose Adenosine Inosine oxidase ribose - 1 - P Pi + O O O NH H O 4 2 N N N HN HN HN

N purine nucleoside O N N NH N NH N N H H 2 phosphorylase 2 H deaminase ribose Xanthine Guanosine Guanine xanthine oxidase O H N HN O O N N H H R. Lyons “Salvage” Pathways for Purine

O N HN O Hypoxanthine- N N N guanine HN H phosphoribosyl Hypoxanthine N N transferase + + O PPi P O O P O OH O P O P OH OH OH Inosine monophosphate PRPP R. Lyons

APRT - phosphoribosyl transferase - performs a similar function with adenine. Adenosine Deaminase Deficiency:

NH2 N O O N N N HN HN N N N N N N HO O Adenosine H deaminase 2- (ADA) Deoxyinosine Hypoxanthine OH H

Guanine Xanthine dAMP

dADP Uric acid

dATP

R. Lyons O N HN Gout: deposition of urate crystals in joints, “ ” N N tophi in cooler periphery H

Hypoxanthine

xanthine Hyperuricemia can be caused by: oxidase

O Accelerated degradation of : N HN

O N N •Accelerated synthesis of purines H H Xanthine •Increased dietary intake of purines

xanthine oxidase Impaired renal clearance of uric acid O H N OH HN H O N N O N N Allopurinol inhibits xanthine oxidase N H H N Uric acid and reduces blood uric acid levels: Allopurinol R. Lyons R. Lyons An 80-year-old man with a 30-year history of gout, this patient had been treated intermittently to reduce his serum urate levels.

The New England Journal of Medicine Lesch-Nyhan Syndrome: Defective HGPRT

• hyperuricemia • spasticity • mental retardation • self-mutilation behavior

O N HN O Hypoxanthine- N N N guanine HN H phosphoribosyl Hypoxanthine N N transferase + + O PPi P O O P O OH O P O P OH OH OH Inosine monophosphate PRPP R. Lyons A defect in APRT does NOT have similar consequences Myoadenylate Deaminase ‘Fills’ the TCA Cycle in Muscle

H2O NH3

myoadenylate deaminase

NH2 O N N N HN N N N N ribose-5-P ribose-5-P

Adenosine monophosphate Inosine monophosphate

Asp, GTP Fumarate

GDP + Pi

To TCA Cycle

R. Lyons

Carbamoyl phosphate synthetase II - a cytoplasmic enzyme…

O - 2- 2ATP + HCO3 + + glutamate + 2ADP + P NH2 C O P i glutamine + H 2O

R. Lyons

…used for pyrimidine synthesis

O O O - O NH - 2 O C C CH HN CH2 NH2 2 C 2- + O O P CH C CH - - O N CO O N CO 2 NH+ 3 CO2 2 H carbamoyl H aspartate orotate phosphate

R. Lyons Orotate is linked to PRPP to form monophosphate:

O

HN O O

O N CO2 H HN HN orotate O N CO2 O N + O O P O P O O P O CO2 O P O P OH OH OH OH OH OH orotidine uridine phosphoribose - 1 monophosphate monophosphate pyrophosphate

R. Lyons Newly-synthesized will be phosphorylated to UDP and UTP, as described for the purine nucleotides.

UTP can be converted to CTP by CTP Synthetase:

O NH2

HN N

O N gln glu O N

O O P O P O P O P O P O P O

OH OH ATP ADP OH OH + + uridine H2O Pi triphosphate triphosphate

R. Lyons Some UDP is converted to dUDP via ribonucleotide reductase.

UDP dUDP dUTP dUMP ribo- nucleotide reductase ATP ADP H2O Pi

R. Lyons The Reaction:

O O

HN HN CH3

O N O N O O P O P O thymidylate synthase

OH H OH H N 5, N10 deoxythymidine monophosphate monophosphate methylene dihydrofolate tetrahydrofolate

DHFR NADH ****

methylene THF NAD+ donor R. Lyons Inhibits :

O O

HN HN CH3

O N O N O O P O P O thymidylate synthase

OH H OH H deoxyuridine N 5, N10 deoxythymidine monophosphate monophosphate methylene dihydrofolate tetrahydrofolate

DHFR NADH **** X Methotrexate methylene THF donor NAD+ R. Lyons Dihydrofolate builds up, levels of THF become limiting, thymidylate synthase is unable to proceed. Follow it with a dose of Leucovorin, a.k.a. formyl-THF. FdUMP Inhibits The Thymidylate Synthase Reaction:

O O F HN HN CH3

O N O N O O P O P O thymidylate synthase X OH H OH H 5-fluorodeoxyuridine N 5, N10 deoxythymidine monophosphate monophosphate (F-dUMP) methylene dihydrofolate tetrahydrofolate

DHFR NADH ****

methylene THF NAD+ donor

R. Lyons Complicated Pathways for Pyrimidine Production:

dCTP CTP UTP dUTP dTTP ***

dCDP CDP UDP dUDP dTDP *** ***

UMP dUMP dTMP ***

de-novo synthesis OMP R. Lyons This figure is primarily a study aid; you do not need to memorize it or reproduce it. The information here merely summarizes material from previous sections. Pathologies of pyrimidine nucleotide biosynthesis:

Orotic acidurea due to OTC deficiency - please review your Urea Cycle notes.

Hereditary orotic acidurea - deficiency of the enzyme that convert orotate to OMP to UMP. Not common. Pyrimidine degradation:

Cytidine deaminase converts cytidine to uridine

O NH2 N HN O N O N cytidine O O HO deaminase HO OH OH OH OH Cytidine Uridine R. Lyons A phosphorylase removes the O

O HN CH 3

HN CH3 O N pyrimidine H O N P phosphorylase + i O + HO O HO O P OH H

(deoxy) OH H deoxyribose-1-phosphate R. Lyons Degradation of the base proceeds (products are unimportant here) can be salvaged as well:

Enzyme: Pyrimidine nucleoside phosphorylases Thymine + deoxyribose-1-phosphate --> thymidine (NOT !)

O

O HN CH3

HN CH3 O N pyrimidine H O N P phosphorylase thymine + i O + HO O HO O P OH H

(deoxy)thymidine OH H deoxyribose-1-phosphate

R. Lyons

Enzyme: - adds the monophosphate back Thymidine + ATP --> thymidine monophosphate

Herpes Simplex Virus carries its own tk gene Certain drugs act via the pyrimidine salvage pathway:

G HSV G HO O thymidine P O O kinase H H H H

Acyclovir AcycloGMP

R. Lyons

O O HN F O HN F O N HN F H pyrimidine O N uridine 5- phosphorylase O kinase O N + HO O O P O HO O P OH H OH H OH H deoxyribose-1-phosphate fluorodeoxyuridine fluorodeoxyuridine monophosphate (FdUMP) R. Lyons 5-FU efficacy depends on rate of degradation vs activation

O O HN F O HN F O N HN F H pyrimidine O N uridine 5-fluorouracil phosphorylase O kinase O N + HO O O P O HO O P OH H OH H OH H deoxyribose-1-phosphate fluorodeoxyuridine fluorodeoxyuridine monophosphate (FdUMP) R. Lyons 5-FU --> --> FdUMP + methylene-THF + Thymidylate Synthase --> inactivation of TS

Degradation (via dihydropyrimidine dehydrogenase, DPD

DPD inhibitors can potentiate 5FU activity mode of action:

O CH3 O HN

N F O O N cytidine pyrimidine HN F dealkylation phoshorylase O deaminase HO O N H OH OH fluorouracil

capecitabine R. Lyons arabinoside (araC) activation and inactivation:

O NH2 NH2 HN N N O N cytidine O N cytidine O N O HO deaminase O kinase O OH HO P O OH OH OH OH OH

Uridine arabinoside Cytosine arabinoside (araC) Cytosine arabinoside monophosphate (INACTIVE) (ACTIVE)

R. Lyons Additional Source Information for more information see: http://open.umich.edu/wiki/CitationPolicy

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