5’-IMP 5’-XMP 5’-GMP GDP GTP dGDP dGTP 5’-AMP ADP ATP dADP dATP

5’-UMP CDP CTP dUTP dCDP UDP UTP dUDP dTDP dCTP dTTP dCMP dUMP dTMP Rapid

Formation of Deoxyribonucleotides Two Component System to Reduce Ribose 1. Enzyme: ribonucleotide reductase 2. Electron transport system: thioredoxin or glutaredoxin Methylation of 5’-dUMP 1. Methylene THF 2. Thymidylate synthase

Ribonucleoside Reductase Tetramer (4 subunits)  2  2 The two  subunits are part of protein R1 The two  subunits are part of protein R2 R1 has the catalytic site R2 has a tyrosine free radical with 2 Fe 3+ 3 cysteines (catalytic site) 2 cysteines (redox active site)

-SH SH HS--SH SH HS- -SH HS- -SH HS- Fe O O O O R1 (  2 ) R2 (  2 ) Redox Site Catalytic Site Binuclear Fe(III) with Tyrosine Free radical Two identical pairs of subunits 2 allosteric sites for ATP, dATP, dGTP, dTTP

Formation of Deoxyribonucleotides OHHO H OHHO H H H H + + H 2 O H Hydride ion H H HO Deoxyribose HO H H H E-X Enzyme E-X-H H E S-H E S S H + E S S E-X-H + E-X

Regeneration of Sulfhydryls Groups in Ribonucleotide Reductase E S S E SH NADPHNADP + FADFADH 2 R SH R S S TR SH TR S S Thioredoxin Reductase Ribonucleotide Reductase

Where are the Electrons NADPH NADPFADH 2 FAD Thioredoxin reductase NADPH NADP TR S S S S H H Glutaredoxin S GR S 2GS-SG 2GSH GR S S H H rNDP reductase rNDP reductase SS rNDP reductase rNDP reductase SSHH rNDP dNDP Glutathione reductase

Catalytic Specificity One enzyme is used for ADP, GDP, CDP, UDP (dTMP is formed via dUMP) Balance is maintained by end products (dNTP’s) Activity sites and Specificity sites ATP increases efficiency for ALL substrates dATP decreases efficiency of all substrates Specificity site maintains balance of the 4 dNTP’s dTTP activates GDP but inhibits UDP, CDP

Post Synthesis modifications 5’-UMPUDP UTP Glutamine Glutamate CTP dUDP dUMP dTMP 5,10-Methylene THF Dihydrofolate N N O O H N N O O H CH 3 Thymine R R Deoxy Thymidine synthesis requires THF Deoxy Thymidine synthesis requires THF

N CH 2 N H2CH2C dUMP dTMP N CH 2 N H N N H Dihydrofolate reductase Dihydrofolate reductase NADPH + H + NADP + Serine Glycine Thymidylate Synthase TS and DHFR are targets of anti-cancer drugs TS and DHFR are targets of anti-cancer drugs H H

N N O O H F 5-fluorouracil PRPP 5-fluoro-dUMP N N O O H CH 2 N NH S- Enzyme H F Suicide Inhibitor Enzyme unable to extract F Enzyme commits suicide by attacking substrate Enzyme commits suicide by attacking substrate (5-fluorodeoxyuridylate) p713

Nucleoside Analogs are the Basis of Anticancer and Antiviral Drugs Why nucleosides? Because they penetrate membranes more readily Because they react with internal kinases Antiviral araA (arabinosyladenine) araC (arbinosylcytosine) Arabinose HO HOCH 2 R R=adenine or cytosine

Aminopterin OH H2NH2N CH 2 H See page 713 CH 3 Methotrexate NH 2 Anti-Leukemia agents blocks deoxy thymidine synthesis Folic Acid Inhibitors of dihydrofolate reductase Inhibitors of dihydrofolate reductase

Dideoxynucleosides 2’3’-Dideoxycytidine 2,3’-Dideoxyinosine H H H H HOCH 2 R N N O O H CH 3 O N=N=N H HOCH ’Azido-2’,3’-dideoxy- thymidine AZT

Quiz 3 Take out a Sheet of paper and number its from 1-7. You will be given 5 minutes to identify by name the factor involved in the following metabolic scheme

PP i N2N2 2 NADPH NADP + 3-phosphohydroxy- pyruvate  -Kg NH 3 Glutamate Glutamine PRPP 5-phospho- ribosylamine 3-phospho- serine