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LECT 35 Deoxy

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					              5’-AMP       ADP    ATP

5’-IMP                     dADP

         5’-XMP            dATP

                  5’-GMP   GDP    GTP


                           dGDP


                           dGTP
5’-UMP          UDP       UTP


                dUDP      CTP    CDP


                dUTP             dCDP
                          dCMP

                  Rapid
                          dUMP   dCTP

         dTTP      dTDP   dTMP
             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
       3 cysteines (catalytic site)
        2 cysteines (redox active site)

R2 has a tyrosine free radical with 2 Fe3+
                                              Redox Site
                  -SH HS-   -SH HS-
   R1 (2)                                2 allosteric sites for
                    SH        SH          ATP, dATP, dGTP, dTTP
                  -SH HS-   -SH HS-
                                             Catalytic Site
                  O              O
             Fe       Fe    Fe       Fe
 R2 ( 2)
                        O             O
                                               Binuclear Fe(III)
                                               with Tyrosine
                                               Free radical
Two identical pairs of subunits
      Formation of Deoxyribonucleotides

Enzyme
E-X        H   H                  H                H
      H                                                + H2O
          HO   OH        HO       +OH     HO
                                                   +
                                  H
E-X-H            S-H
               E                          H
                 S-H            S
                              E
                                SH        S
                                        E
                                          S

                                      E-X-H
                       H      H                    H   Hydride ion
          E-X +
                    HO      H                 HO   H
                       Deoxyribose
Regeneration of Sulfhydryls Groups in
    Ribonucleotide Reductase
                                        Ribonucleotide
               SH                S        Reductase
           E                 E
               SH                S
               S             SH      Thioredoxin
            TR            TR
               S             SH

             SH            S
           R             R
             SH            S
                                        Thioredoxin
                                          Reductase
            FAD          FADH2


            NADPH      NADP+
    Where are the Electrons                     dNDP
                                    rNDP
                                    reductase
NADPH          FAD      TR SH
                           SH
                                     S     S
 NADP          FADH2    TR S
                           S
          Thioredoxin
          reductase
                            SH      rNDP
NADPH          2GS-SG    GR SH
                                    reductase

                             S       HS    SH
 NADP          2GSH      GR S
 Glutathione                                    rNDP
 reductase              Glutaredoxin
          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’-UMP       UDP                UTP
                        Glutamine
       O                                     Deoxy Thymidine

HN               dUDP Glutamate              synthesis requires
                                                   THF
                                    CTP
O      N         dUMP
       R                5,10-Methylene THF
      O
HN         CH3           Dihydrofolate

O                dTMP
      N                  Thymine
      R
          dUMP                           dTMP
                          Thymidylate
                          Synthase
            N      CH2                   N       CH2
         H2C       N
                                                 N
                                                 H
                Glycine
                                             NADPH + H+
                  Serine           H         NADP+
                           N       CH2         Dihydrofolate
                           H
                                   N           reductase
                                   H

      TS and DHFR
are targets of anti-cancer drugs
                                Enzyme commits suicide
            O                    by attacking substrate
       HN        F      PRPP
                                    5-fluoro-dUMP
       O    N                      (5-fluorodeoxyuridylate)

       5-fluorouracil
                                         N     CH2
                                  O      CH2
                           HN                   NH
                                        F
                                        H
                           O      N
 Suicide Inhibitor                       S- Enzyme


p713                           Enzyme unable to extract F
 Nucleoside Analogs are the Basis of Anticancer and
                  Antiviral Drugs

Why nucleosides?
Because they penetrate membranes more readily

Because they react with internal kinases
                                       HOCH2         R
  Antiviral
                                                HO
araA (arabinosyladenine)
                                           HO
araC (arbinosylcytosine)
                                           Arabinose
                                      R=adenine or cytosine
   Folic Acid                         See page 713

  H2N       N      N   H
                                            O      O
                                                  CO
        N                                       H
                   N   CH2   N              C   N C H
                             H
                             CH
            OH 2
            NH                    3               H
                                                  C2
                                                     H
                                                     C2
                                                     O
                                                     CO
Aminopterin                Methotrexate
                                      Inhibitors of dihydrofolate
Anti-Leukemia agents
                                            reductase

            blocks deoxythymidine synthesis
Dideoxynucleosides
                                  HOCH2         R
2’3’-Dideoxycytidine

2,3’-Dideoxyinosine                    H    H

                                       H    H
                      O
              HN          CH3

             O        N
        HOCH2
                O           3’Azido-2’,3’-dideoxy-
                                  thymidine
      - +
      N=N=N         H               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
                      1
      N2           2 NH3

                          -Kg
                           2
           NADPH
                                           PRPP
                                             5
                              1
                             NH
           NADP+                  3

                   3                  Glutamine
                                           4
              Glutamate
3-phosphohydroxy-
   pyruvate                              3
                                 Glutamate        PPi
    3-phospho-
            7
     serine
                                        5-phospho-
                                              6
                   -Kg
                     2                  ribosylamine

				
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