Chp6 by 2eV8w9

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									 The Organic Chemistry of
Enzyme-Catalyzed Reactions

        Chapter 6

       Substitutions
                                      SN1
        Reactions catalyzed by farnesyl diphosphate
                         synthase

                               -PPi
                                                             geranyl
PPO          +   PPO                   PPO
                       6.2                    6.3
                                                             diphosphate
      6.1
isopentenyl DP   dimethylallyl DP
                                                    -PPi
                                        PPO

                                                             farnesyl
                                                             diphosphate
                                PPO
                                              6.4

                                                           Scheme 6.1
  Hammett study supports carbocation intermediate
                 PPO
                            F
                                  6.5


Km same as geranyl DP, but kcat 8.4  10-4 times
that with geranyl DP

 Therefore, it binds as well as geranyl DP, but is converted to product
 at a much slower rate, supporting an electron-deficient intermediate
 (such as a carbocation).
      Model Studies to Test Mechanism
1. Solvolysis (carbocation mechanism)
    rate with X = F is 4.4 x 10-3 times rate with X = H
2. SN2 rate with X = F is 2 x faster than when X = H

                   O
              H3C S O

                   O        X
                                6.7
    The enzymatic reaction is 8.4 x 10-4 times
    slower when X = F compared to X = H

         Therefore carbocation mechanism
  Further Support for Carbocation Mechanism

             CH2F                      CHF2               CF3

   PPO                        PPO                 PPO

              6.8                       6.9                6.10
Relative
           1.75  10-2              1.90  10-6         3.62  10-7
rate
                compared with geranyl DP (CH3)
                    Km values similar to geranyl DP

     Rates correlate with nonenzymatic solvolysis
     for fluorinated methanesulfonates relative to
     geranyl DP (carbocation mechanism)
                  Carbocation Mechanism (SN1) for
                  Farnesyl Diphosphate Synthase
                          +                + PPi
PPO               R                  R
                              6.11
R = Me (6.2)
R = C5H11 (6.3)



                      +
PPO                                      PPO       +   R   PPO   R
                          R
                                               H
                                          B:


Scheme 6.2
    Stereochemistry of Farnesyl Diphosphate
                   Synthase
               syn addition/elimination


                              PPO

                                         6.21


                                    si
             PPO


                    HR   HS
               B:
                      6.20



Figure 6.1
      Sesquiterpenes Biosynthesized from Farnesyl DP
         Reaction catalyzed by pentalenene synthase
                                                     309His                         309His




                                                      NH                                NH
                                                 N                           N
                                                                         H
                                             H




                                                                                 6.22
PPO
  Mg2+
                                    309His
                                                              humulene                       309His


                                     NH
                                N                                                             NH
                                                                                      N
                        H   H                         H                      H
             H


                                                                                  H



                                                                         6.23
           6.24

      pentalenene
                                                                  Scheme 6.5
From the Crystal Structure of Pentalenene Synthase
    Stabilization of carbocation intermediates by
 active-site phenylalanine and asparagine residues

                   cation- interaction

                                              NH2
                                 
                                   O      C
                                          219Asn




           Phe77      carbocation
                      stabilization
  Figure 6.2
                           SN1/SN2

      Reaction catalyzed by phosphorylases

                    O                   O
R O   R'   +   -O   P O-             RO P    O-   +   R'OH
                    OH
                                        O-



Scheme 6.6
          Reaction Catalyzed by Disaccharide
                   Phosphorylases
      OH
                                            OH
            O         another sugar                O
     OH                                    OH          OPO3= +   H18OR
                          +   Pi
HO          18O                       HO
                  R                                              6.27
          HO                                    HO
     6.25                                   6.26




 Scheme 6.7
      Stereochemistry of the Reactions Catalyzed by
              Disaccharide Phosphorylases


                           C-1 Configuration       C-1 Configuration
                            of Disaccharide    of Phosphorylated Product
cellobiose phosphorylase                                 
maltose phosphorylase                                                    inversion
sucrose phosphorylase                                                    retention
             Two Mechanisms for Inversion
            SN2 versus stereospecific SN1 reaction
                                                      OH
                                                               OPO3H-
                                          SN2              O
                                                     OH
             OH
                      SN1 S 2
                           N                    HO
                  O
                  :             -OPO H-                   HO
                                    3
            OH
                                                      OH
       HO              OR                                  +     -OPO H-
                 HO                       SN1              O         3
                                                     OH
       Scheme 6.8                               HO
                                                          HO

No partial exchange reactions with cellobiose or maltose
phosphorylases (consistent with SN2)
With sucrose phosphorylase, [14C]fructose is incorporated into
sucrose in presence of unlabeled sucrose and in absence of Pi
Suggests double SN2 displacement
                Covalent Catalysis
                 sucrose phosphorylase
                     OH
                                    OH
                        O
                                         O
          HO                              HO
                             O
           HO      HO                               OH
                                   HO
                glucosyl                fructosyl

                            6.28

14C in glucosyl part                       14C in fructosyl part
gives 14C-protein                          gives no 14C-protein
(quench at low pH)
 Again consistent with a double displacement mechanism
   Experiments to Identify Active Site Residue (X)
                           MeOH
1. [14C] glucosyl enzyme          6.29 (R = H), not 6.29 (R = Me)
                                   (glucose)
               OH                             OH
                 O                              O
                       X                                  OR
     HO                             HO
          HO      OH                     HO          OH
                                              6.29

2. [14C] glucosyl enzyme very sensitive to base
                                                               O
                           NH2OH
3. [14C] glucosyl enzyme            6.29 (R = H) +
                                                               NHOH
                  Therefore X is Glu or Asp
Disaccharide Phosphorylase Reactions
 Involving an Active-site Carboxylate

       OH             O                                            O
                                                     OH
            ..   O-   SN2                                    O
            O                                            O
      OH                    SN2              OH
                                                                   -OPO H-
                                                                       3
 HO               OR                 HO
           HO                                        HO
                                                 6.31                            SN2
                  H
           B+
                                         a
                          SN1
                                                         O
                                  OH             -
                                                                             OH
                                             a       O
                                     +
                                     O                       b                   O
                                OH                                          OH
                                                         -OPO H-
                                                             3
                          HO                         b                 HO             OPO3H-
                                    HO                                           HO
                                6.32

  Scheme 6.9
        Two Mechanisms for Reactions Catalyzed by
        -Glycosidases--Hydrolysis of Disaccharides
                  acid                                                                          Scheme 6.10
                                           O                 OH
                       OH        H O                          O       -
                        O                         HO                  O       O          SN2 (inversion)
        HO                   O             -ROH    HO
A            HO              R
                       HO H OH                           HO       OH          (General acid/base mechanism)
                                                              HO          O
                             -O
                                       O

             base

             acid
                                                                -             O
                  OH        H O        O                OH    H O                         OH HO        O
                                               HO        O                                 O OH
      HO           O
                            OR
                                   -ROH
                                                 HO           OH                  HO                   double
B                                                                                   HO
        HO                                                                                             SN2
              HO                                      HO O                                 HO   -O
                       -O                                         O                                  O (retention)
                             O
nucleophile                                                                                            (Covalent)
                                       Mutation to Ala: kcat 107-fold lower
    Two active site                    Add in N3- to replace the carboxylate nucleophile:
    carboxylic acids                   kcat only 102-fold lower (-azide forms)
      Differentiation of SN2 from SN1
            for -Glycosidases
         OH
                                 X = F or     really good
              O    X                          leaving groups
                             O          NO2
HO
 HO           F
                                 NO2
       6.33
              more electronegative than OH
              destabilizes an oxocarbenium ion intermediate

        SN1 reaction slower than glycoside
        SN2 reaction faster than glycoside

         Covalent adduct stabilized
                     Reaction of 6.33-Inactivated
                      -Glucosidase with 6.35

            OH                        OH                                              OH
             O                         O                                               O    Ph
  HO                                            Ph                     OH
    HO                    +                                             O
            F
                              HO
                               HO          OH                                  O HO    OH
                 O                                             HO
                      O             6.35                         HO      F

         6.34                                        OH
                                                                             6.36

   after isolation                                     O   X             Scheme 6.11
   (Glu-358)                         HO
                                      HO               F

                                                6.33

6.36 formed from 6.34 at same rate as from 6.33                       2nd step must be rds
           Therefore 6.34 is a kinetically competent intermediate,
           consistent with SN2 mechanism followed by SN1
        Both SN2- and SN1-like Character of
                  -Glucosidase
                                                   H B   ‡
        OH        B                       OH                             OH
             H                             O       OR                                B–
 HO      O                        HO                               HO
             OR       SN2-like                                               O   H
  HO                               HO                              HO
        OH                               HO
                                               O–                       HO           O H
             O–
         O                                O                                  O
                                                                        O


substitution of                                                          SN1-like
Glu-358 by Asn                                                                            ‡
or Gln - inactive                                                            H B
                                    OH         B                    OH OH
by Asp - 2500x              HO       O    H                          O+
                                          OH                 HO
slower                       HO                               HO
                                    OH
                                         O–                        HO O–
                                    O                               O

       Scheme 6.12
                                               SN2
              Two mechanisms for epoxide hydrolase
General base mechanism
                            O                                OH
A   Enz B:                             EnzBH       +
                   H   OH
                                                       HO

Nucleophilic (covalent) mechanism
                                                        OH               O
          O            O                   O                                               OH
B                                                                  Enz       O–   +
    Enz       O–                     Enz       O
                                                                                      HO
    B                                                             BH+
                                B:                                           Scheme 6.14
                                       H OH



 Single-turnover experiment in H218O - no 18O in glycol
 Enzyme labeled with 18O in active site Asp gives 18O glycol
          Consistent with covalent catalytic mechanism
                     Further Evidence for Ester Linkage
             Covalent intermediate isolated during reaction
                   catalyzed by epoxide hydrolase
    H+
    O 3                                         OH 3H                                          OH 3H
       H
                               CO2Me                                           CO2Me                             CO2Me
                                                O                                              HO
     O-                                     O                    6.39
O                6.38
                                                    333
                                                               quenched (AcOH)
    Asp333                              H OH Asp
                                   N
                                                               precipitated (acidic acetone)
                                 NH



             OH 3H                                         OH 3H
                                                                                                       OH 3H
                                            LiAlH4                                             NaOH
                                       OH                                              CO2Me
          HO                                               O                                           HO                CO2H
                                                     O
                        6.40                                                                                   6.41
                                                          Asp333    isolated

Scheme 6.15
A Catalytic Antibody-catalyzed 6-Endo-tet
              Ring Closure
        Baldwin’s rules predict 5-exo-tet

                                    a                                    H
  HO                                                                HO
              6-endo-tet                           5-exo-tet
                            O              a
                 b                                     a                     O
        O                       b           :O                           H
                                          b                    Ar
 Ar    6.43                                  H
                           Ar
                                        6.42
obtained with a                                  1.8 kcal/mol lower in
catalytic antibody                               energy in solution
(anti-Baldwin product)


Scheme 6.16
                         SN2

Reaction catalyzed by isochorismate synthase

  COO-                               COO-
                                            18OH
                     isochorismate
                        synthase
          O   COO-    Mg++ H218O              O    COO-
  OH
                                       6.45
   6.44


Scheme 6.18
 SN2 Mechanism for Isochorismate Synthase

        COO-        H       :B                                           ‡
                                    -OOC       O                             COO-
                18O     H                                                       18OH
                                                                  COO-

                O       COO-                                                        O   COO-
                                               H
        OH                                 O            O18
 B+ H                                H             2+         H
         6.44                                  Mg

                                               6.46

                                 all axial conformation
Scheme 6.19
Reaction Catalyzed by Anthranilate Synthase

    CO2-                        CO2-                  CO2-
                       NH3             NH3                   NH3+

                       -H2O
            O   CO2-                   O     CO2-
    OH                                                6.48
                                  6.47
     6.44
                              synthesized -
                              kinetically competent
Scheme 6.20                   intermediate
Reaction Catalyzed by p-Aminobenzoic Acid (PABA)
                    Synthase

 Scheme 6.21                     synthesized -
                                 kinetically competent
      CO2-                            CO2-                CO2-
                          NH3
                          -H2O
              O    CO2-                        O   CO2-
                                     +NH
      OH                                   3              NH3+
       6.44                                6.49           6.50


                  reaction different from others
  Synthesized as TS‡ Mimics of the 3 Enzymes
         (in the all-axial conformation)
isochorismate synthase   anthranilate synthase PABA synthase
     CO2-                   CO2-
                                   +            CO2-
            OH                     NH3                 OH

            O    CO2-              O     CO2-          O    CO2-
     OH                     OH                  +NH
                                                  3

     6.51                   6.52                6.53

   All 3 compounds competitive inhibitors of respective
   enzymes; bind tightly to isochorismate and
   anthranilate synthases, but weakly to PABA
   synthase (different mechanism)
Nucleophilic Aromatic Substitution (SNAr)

           Glutathione (GSH)
              COO-           O
                         H
          +              N
        H3N                       N   COO-
                                  H
                     O
                             SH
                     6.57

         g-glutamylcysteinylglycine
                                  SNAr
  Reaction catalyzed by glutathione S-transferase
   Tyr
         O                    Tyr
         H                            O                 Scheme 6.23
                                       H   _
    GS       X   O                    X  O               GS
                             GS              _
                 N O-                    N O                  NO2
                 +    slow               +       fast
                                                               +    HX

             Y                    Y                      Y


Hammett study
     = +1.2 for GSH
                                             therefore carbanionic
      = +2.5 for g-Glu-Cys
   rate X = F > X = Cl
Glutathione S-transferase-Catalyzed Reaction
  of Glutathione with 1,3,5-Trinitrobenzene

                                                GS     H
       O2N                 NO2
                                          O2N               NO2
                                 GS-


                 N+
                      O-                             NO2-
             O
                                                     6.58
Scheme 6.24                      observed spectroscopically

                                       Meisenheimer complex
               Electrophilic Substitution
           (Addition/Elimination Mechanism)
 Reaction catalyzed by 5-enolpyruvylshikimate-
        3-phosphate (EPSP) synthase
            CO2-                                           CO2-


                        +                                                     +   Pi
=O                          =O PO          CO2-
     3PO           OH                             =O PO            O   CO2-
                              3                     3
            OH                      6.61                   OH
           6.60                                            6.62

shikimate-3-P                   PEP
                                                          EPSP

                                                                  Scheme 6.29
Herbicide Glyphosphate (Roundup™)
       inhibits EPSP synthase

                +
         -OOCCH NH CH PO =
               2  2  2  3

               6.63
     4 Possible Mechanisms for EPSP Synthase
1) Concerted
                                                       :B
                                                H
                  H B+
     ROH                                        CH2                    - Pi
                          addition
      :




                                           +
       -O         OPO3=                   RO        OPO3=         elimination        RO          CO2-
            2C
                                               CO2-                                       6.62
                                          H
                                     B:


2) Stepwise

                 H B+
                                                                                                        B:
                                                            :

                                                            ROH        +                                      H
                                           +                           RO        OPO3=
    -O C         OPO3=        -O C                                                                                CH2        6.62
      2                         2              OPO3=
                                                                       H      CO2-                           RO
                                                                                                                  +
                                                                  B:                                                  CO2-




 Scheme 6.30 (continued on next slide)
4 Possible Mechanisms for EPSP Synthase (continued)
      3) Covalent-concerted (a) and   4) Covalent-stepwise (b)



                     H B+                              :B                                H B+
                                                   H
                                              H2C
                                                  b           b
          X-                                X       OPO3=                    X
           -O        OPO3=                         _      stepwise                          _
                2C                                                                       CO2
                                           a    CO2
                                  RO H
                                       a              concerted
                                   B:
                                                  H                              CH3
                                          H2C            :B                                 _
                                                        _                X               CO2
                                      X           CO2                            +

                                           RO




                                                                               :
                                                                             ROH



                                                                                           :B
                                                                                     H
                                                                         H2C
                                                       _                                  _
                                      RO          CO2                X               CO2
                                           6.62                          RO          H
 Scheme 6.30                                                                               :B
          Isolated by Et3N Quench
                       CO2-

                                  CH3
               =O PO
                 3            O     OPO3=
                       OH         CO2-

                       6.64

         Incubated with EPSP synthase -
         kinetically competent intermediate

Therefore not covalent mechanisms (3 or 4)

Kinetic analysis indicates only one intermediate
detected; therefore mechanism 1 proposed
              Evidence for Stepwise Mechanism 2
               EPSP synthase-catalyzed reaction of
            shikimate-3-phosphate and (Z)-3-fluoroPEP

           CO2-                                         CO2-                            CO2-
                             F                                                                      F
                                                                    CH2F                                       + Pi
=O                     =O           COO-   =O                         CO2-   =O
                                                                                  3PO           O       CO2-
     3PO          OH        3PO                 3PO             O
                                                                    OPO3=
           OH                                           OH                              OH
                                 6.65
           6.60                                          6.66                            6.67
                                                      isolated
Scheme 6.31                                                                  does not give 6.66
                                                                             (reverse reaction)


 Not much carbocation character in the addition step,
 but high carbocation character in elimination step
           Carbocation Character in the Reaction
               Catalyzed by EPSP Synthase

            CO2-                           CO2-

                       CH3                            CH3
                                                               EPSP
=O                 O     CO2-                           CO2-
     3PO
                                =O                O
                                     3PO
                       OPO3=
            OH                             OH
            6.68                           6.69


Scheme 6.32
To Determine Stereochemistry of Tetrahedral
               Intermediate
                phosphonate (stable)
         CO2-                           CO2-

                    CH3                            CH3
 =O PO
   3            O     PO3=      =O PO
                                  3            O     CO2-
         OH         CO2-                OH          PO3=
         6.71                           6.72
     Ki = 15 nM*                   Ki = 1130 nM
   (suggests this
  stereochemistry)
To make a stable phosphate, put in an electron
withdrawing group

                         CH2F, CHF2, CF3

         CO2-                             CO2-
                    X                                X
 =O PO
   3            O    OPO3=        =O PO
                                    3            O    CO2-
         OH         CO2-                  OH         OPO3=
         6.73                             6.74
                                 more potent inhibitor
                             (opposite stereochemistry as
                             the phosphonate analogues)
                                               MurA
      (Bacterial cell wall peptidoglycan biosynthesis)
             Similar reaction to EPSP synthase
       Reaction catalyzed by uridine diphosphate-N-
     acetylglucosamine enolpyruvyl transferase (MurA)
          OH                        O                                         OH
          O                                                                                            O
HO                             NH                                             O
                                                                  HO                           NH
 HO       NH    O   O      O        N                       -Pi     O
                                                                              NH    O   O      O       N
      O     O   P O P O        O         +   =O PO   CO2-
                                               3                          O     O   P O P O        O
                O-  O-                                             COO-
                                                                                    O-  O-
                 6.75     HO        OH
                                                                                    6.76      HO       OH


opposite results                                 Scheme 6.34
          Kinetics suggest tetrahedral noncovalent intermediate
                [14C]PEP or [32P]PEP gives labeled enzyme
                NMR with [2-13C]PEP shows phospholactyl enzyme
                adduct (kinetically competent)
    One Possible Mechanism for the Reaction
              Catalyzed by MurA
                                                 noncovalent
                   covalent                      intermediate
                 intermediate                             OH                 OH
B   H                    OPO3   =                           O
         OPO3=                                     HO                          O
                                    UDP-GlcNAc                      HO
         CO2-            CO2-                       O
                                                       - HN O-UDP    O               + Pi
                     X                              CO2                     HN O-UDP
    X-                                                        O                  O
                                                  OPO3=              CO2-
                     6.77
                                                        6.78                6.79
          phospholactyl enzyme
          kinetically competent

Scheme 6.35
              Further Evidence for Covalent and
                  Noncovalent Intermediates
                  Inactivation of MurA by (E)- and (Z)-3-
                                 fluoroPEP
                                                  OPO3=                      OH
H          OPO3=        F          OPO3=                                      O
                                           FCH2     CO2-          HO
                   or                                              O
                                                           -O C              NH
F          CO2-         H          CO2-           X          2
                                                                     OPO3=      O UDP
    6.80                    6.65                                             Ac
                                                              FCH2
                                                  6.81                  6.82
Scheme 6.36                                covalent               noncovalent
                                           (stable)

    Kinetics suggest that 6.82 does not come from 6.81
                                Branching Mechanism
             More consistent mechanism for the reaction
                         catalyzed by MurA

                            +O—PO =
                                                       OPO3=           H            CO2-   H          CO2-
H           OPO3=                3       ROH                   - Pi   H
                                               H3C      CO2-
H                    H3C                                                H           O+
            CO2-                CO2-             RO:                                       H          OR
        H                                                               B-          R          6.79
                           6.83                      6.84
    B
                       X-                  noncovalent                       6.85
                                           intermediate
                      CH3       OPO3=

                                  CO2-
                                                                               Scheme 6.37
                            X


                            6.86
                      covalent
                    intermediate
                                                        H            OPO3=              F                OPO3=
  Determination of
                                                          F          CO2-                H               CO2-
the Stereochemistry             From crystal                  6.80                               6.65

  of the Reaction               structure [2R];                             MurA
                                therefore ROH                               ROH/D2O
 Catalyzed by MurA              addition is 2-si               OR                         F
                                                                                                        OR
                                                         H       CO2-                                    CO2-
                                                        D                   [2R]         D                      [2R]
                                (top) (2-re in
    Scheme 6.38                                           F OPO3                           H        OPO3
                                PEP)                       6.87E                                 6.87Z

                          H        OR        -UDP-GlcNAc                 alkaline                   -UDP-GlcNAc
                                                                         phosphatase
                      F
                                                              O                                    O
                                     OPO3=           H
                          D       CO2-
                                                                                            F            fluoropyruvate
                                                    D             CO2-                                  CO2-
                          3R                                                             D
                                  2R
                                                        F 6.88E                              H 6.88Z
                             6.87E
                                                                         pyruvate
                                                                         carboxylase

                                 fluorooxaloacetate                      (retention)              O
                                                   -OOC
                                                              O                        -OOC

Analyzed for H or D by 19F NMR                       D            CO2-                   H              CO2-
                                                         F                                   F
Therefore addition of D+ is to 3-re                  malate
                                                     dehydrogenase
face (bottom), which is called si
                                                                                                   OH
with PEP; addition of ROH is to fluoromalate OH
                                       -OOC                     -OOC                                      H
                                                                                                              fluoromalate
                                                  H
2-si (top), which is 2-re in PEP         D      CO2-               H                                     CO2-
                                          F                          F
         Anti addition                      6.89E                                                 6.89Z
                       Stereochemistry of the Reaction
                             Catalyzed by MurA

                 OH
                  O
            HO                re                                                                                                      re
             O
  B:       H     NH                                                   OH
                 Ac O UDP                                              O                                                   OH
                                                            HO
                                                                                                                            O
                                   anti                       O                        syn                       HO
       H              OPO3=   si   addition
                                                    H
                                                                          NH           elimination                 O                  si
                                                                  OPO3=        O UDP                 H
       H              COO-                          H                     Ac                                               NH
                                                        H     COO-                                                         Ac
                                                                                                                              O UDP
                                                                                                     H            COO-
           H                                    S
       S                                                                                                     H
                                              115Cys                                                     S
   115Cys
                                                                                                     115Cys        HPO4=



                                                                               Not concerted
Scheme 6.39
Electrophilic Aromatic Substitution
  Friedel-Crafts reaction (alkylation)

                       R' H   -X     R'


      R' X   AlCl3       +


 R                       R           R


Scheme 6.40
Enzymatic Friedel-Crafts Reactions
     (alkylation in nature)
COO-                                               COO-

                   CH3
         +
                                 OPP                                   8   CH3
             CH3           8                              H
O                                                  O          :B
    H                                     B+   H
    B:



                                                   COO-

                          coenzyme Q
                           vitamin K
                         other quinones
                                                                   8   CH3
                                                   OH

Scheme 6.41
  Electrophilic Heteroaromatic Substitution
              porphobilinogen deaminase
                                       A = acetate
                                       P = propionate
              COOH
HOOC                      A       P   A        P A       P A       P


         N                    N           N          N         N
                              H           H          H         H
 H2N     H           HO
       6.90                                   6.91



                                      porphyrins                   corrins


                                          heme           coenzyme B12
                            1) E2' (1,6-elimination)
 Three Possible Mechanisms                         concerted
                                       A           P                                 P                 A       P
for the Reaction Catalyzed by                                        A
                                                                                         Nu
 Porphobilinogen Deaminase                 N                                 N                             N
                                                                                                  Nu       H
                                    NH3+   H

                                               B




                            2) E1cB

                                      A        P
                                                       anionic   A               P
                                                                                         Nu
                                           N                             N
                                    NH3+                    NH3+
                                           H

                                               B



                            3) E1
                                                   cationic
                                       A           P             A               P            A        P
      Scheme 6.43
                                           N                             N                        N
                                    NH3+   H                                                      H
                                                                         H
                               Substrate Analogues
 A           P          A           P          A           P          A          P          A          P

                 H3C                    F3C                    H3C                   F3C
       N                      N                     N                     N                      N
                              H                     H                     H                      H
NH2    CH3             NH2                    NH2                    OH                    OH
      6.92                   6.93                   6.94                  6.95                  6.96




substrate                                     excellent                    not substrates
(but no tetrapyrrole                          substrates
formed-only tripyrrole)
                                          Consistent with E1 mechanism
 Therefore E2 and
 E1cB unlikely
          Cation Mechanism Most Reasonable
                         Carbocation mechanism for
                         porphobilinogen deaminase
                               A        P


                                   N
               A           P       H



      A   P          N         A        P           A      P    A        P         A             P    A       P
                     H
              NH2
                    -NH3                                 +N         N
      N                            N+                                                       N             N
                                   H                      H H       H
  NH2 H                                            NH2
                                                                                            H             H
                                                                                  NH2
+ H                                                        B:
B


                                                                    A         P   A             P A       P   A       P

                                            6.91
                                                                         N+             N             N           N
                                                                         H              H             H           H
                                                          HO    H
                                                                    :B
Scheme 6.44

								
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