The Organic Chemistry of EnzymeCatalyzed Reactions Chapter 6 Substitutions

1
The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 6 Substitutions
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Reactions catalyzed by farnesyl diphosphate
synthase
SN1
geranyl diphosphate
isopentenyl DP
dimethylallyl DP
farnesyl diphosphate
Scheme 6.1
Hammett study supports carbocation intermediate
3
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).
4
Model Studies to Test Mechanism

• 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

The enzymatic reaction is 8.4 x 10-4 times slower
when X F compared to X H
Therefore carbocation mechanism
5
Further Support for Carbocation Mechanism
Relative rate
1.75 ? 10-2
1.90 ? 10-6
3.62 ? 10-7
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)
6
Carbocation Mechanism (SN1) for Farnesyl
Diphosphate Synthase
Scheme 6.2
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Stereochemistry of Farnesyl Diphosphate Synthase
syn addition/elimination
si
Figure 6.1
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Reaction catalyzed by pentalenene synthase
Sesquiterpenes Biosynthesized from Farnesyl DP
humulene
pentalenene
Scheme 6.5
9
Stabilization of carbocation intermediates by
active-site phenylalanine and asparagine residues
From the Crystal Structure of Pentalenene Synthase
cation-? interaction
carbocation stabilization
Figure 6.2
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Reaction catalyzed by phosphorylases
SN1/SN2
Scheme 6.6
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Reaction Catalyzed by Disaccharide Phosphorylases
Scheme 6.7
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Stereochemistry of the Reactions Catalyzed by
Disaccharide Phosphorylases
inversion
retention
13
SN2 versus stereospecific SN1 reaction
Two Mechanisms for Inversion
Scheme 6.8
No partial exchange reactions with cellobiose or
maltose phosphorylases
(consistent with SN2)
With sucrose phosphorylase, 14Cfructose is
incorporated into sucrose in presence of
unlabeled sucrose and in absence of Pi
Suggests double SN2 displacement
14
Covalent Catalysis
sucrose phosphorylase
14C in glucosyl part gives 14C-protein (quench at
low pH)
14C in fructosyl part gives no 14C-protein
Again consistent with a double displacement
mechanism
15
Experiments to Identify Active Site Residue (X)
MeOH
1. 14C glucosyl enzyme 6.29 (R H),
not 6.29 (R Me)
(glucose)
2. 14C glucosyl enzyme very sensitive to
base 3. 14C glucosyl enzyme 6.29 (R
H)
NH2OH

Therefore X is Glu or Asp
16
Disaccharide Phosphorylase Reactions Involving an
Active-site Carboxylate
Scheme 6.9
17
Two Mechanisms for Reactions Catalyzed by
?-Glycosidases--Hydrolysis of Disaccharides
acid
Scheme 6.10
SN2 (inversion)
(General acid/base mechanism)
base
acid
double
SN2 (retention)
nucleophile
(Covalent)
Mutation to Ala kcat 107-fold lower Add in N3-
to replace the carboxylate nucleophile kcat only
102-fold lower (?-azide forms)
Two active site carboxylic acids
18
Differentiation of SN2 from SN1 for ?-Glycosidases
really good leaving groups
more electronegative than OH
destabilizes an oxocarbenium ion intermediate
slower than glycoside
SN1 reaction SN2 reaction
faster than glycoside
Covalent adduct stabilized
19
Reaction of 6.33-Inactivated ?-Glucosidase with
6.35
after isolation (Glu-358)
Scheme 6.11
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
20
Both SN2- and SN1-like Character of ?-Glucosidase
substitution of Glu-358 by Asn or Gln - inactive
by Asp - 2500x slower
Scheme 6.12
21
Two mechanisms for epoxide hydrolase
SN2
General base mechanism
Nucleophilic (covalent) mechanism
Scheme 6.14
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
22
Covalent intermediate isolated during reaction
catalyzed by epoxide hydrolase
Further Evidence for Ester Linkage
quenched (AcOH)
isolated
Scheme 6.15
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A Catalytic Antibody-catalyzed 6-Endo-tet Ring
Closure
Baldwins rules predict 5-exo-tet
obtained with a catalytic antibody (anti-Baldwin
product)
1.8 kcal/mol lower in energy in solution
Scheme 6.16
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Reaction catalyzed by isochorismate synthase
SN2?
Scheme 6.18
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SN2? Mechanism for Isochorismate Synthase
all axial conformation
Scheme 6.19
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Reaction Catalyzed by Anthranilate Synthase
synthesized - kinetically competent intermediate
Scheme 6.20
27
Reaction Catalyzed by p-Aminobenzoic Acid (PABA)
Synthase
synthesized - kinetically competent
Scheme 6.21
reaction different from others
28
Synthesized as TS Mimics of the 3 Enzymes (in
the all-axial conformation)
isochorismate synthase
anthranilate synthase
PABA synthase
All 3 compounds competitive inhibitors of
respective enzymes bind tightly to isochorismate
and anthranilate synthases, but weakly to PABA
synthase (different mechanism)
29
Nucleophilic Aromatic Substitution
(SNAr) Glutathione (GSH)
g-glutamylcysteinylglycine
30
Reaction catalyzed by glutathione S-transferase
SNAr
Scheme 6.23
Hammett study ? 1.2 for GSH 2.5 for
?-Glu-Cys rate X F gt X Cl
therefore carbanionic
31
Glutathione S-transferase-Catalyzed Reaction of
Glutathione with 1,3,5-Trinitrobenzene
Scheme 6.24
observed spectroscopically
Meisenheimer complex
32
Reaction catalyzed by 5-enolpyruvylshikimate-3-pho
sphate (EPSP) synthase
Electrophilic Substitution (Addition/Elimination
Mechanism)
PEP
shikimate-3-P
EPSP
Scheme 6.29
33
Herbicide Glyphosphate (Roundup) inhibits EPSP
synthase
34
4 Possible Mechanisms for EPSP Synthase
Scheme 6.30 (continued on next slide)
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4 Possible Mechanisms for EPSP Synthase
(continued)
Scheme 6.30
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Isolated by Et3N Quench
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
37
EPSP synthase-catalyzed reaction of
shikimate-3-phosphate and (Z)-3-fluoroPEP
Evidence for Stepwise Mechanism 2
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
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Carbocation Character in the Reaction Catalyzed
by EPSP Synthase
Scheme 6.32
39
To Determine Stereochemistry of Tetrahedral
Intermediate
phosphonate (stable)
Ki 15 nM (suggests this stereochemistry)
Ki 1130 nM
40
To make a stable phosphate, put in an electron
withdrawing group
CH2F, CHF2, CF3
more potent inhibitor (opposite stereochemistry
as the phosphonate analogues)
41
Reaction catalyzed by uridine diphosphate-N-acetyl
glucosamine enolpyruvyl transferase (MurA)
MurA (Bacterial cell wall peptidoglycan
biosynthesis) Similar reaction to EPSP synthase
Scheme 6.34
opposite results
Kinetics suggest tetrahedral noncovalent
intermediate 14CPEP or 32PPEP gives labeled
enzyme NMR with 2-13CPEP shows phospholactyl
enzyme adduct (kinetically competent)
42
One Possible Mechanism for the Reaction Catalyzed
by MurA
noncovalent intermediate
covalent intermediate
phospholactyl enzyme kinetically competent
Scheme 6.35
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Inactivation of MurA by (E)- and (Z)-3-fluoroPEP
Further Evidence for Covalent and Noncovalent
Intermediates
Scheme 6.36
covalent (stable)
noncovalent
Kinetics suggest that 6.82 does not come from 6.81
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More consistent mechanism for the reaction
catalyzed by MurA
Branching Mechanism
noncovalent intermediate
Scheme 6.37
covalent intermediate
45
Determination of the Stereochemistry of the
Reaction Catalyzed by MurA
From crystal structure 2R therefore ROH
addition is 2-si (top) (2-re in PEP)
2R
2R
Scheme 6.38
fluoropyruvate
(retention)
fluorooxaloacetate
Analyzed for H or D by 19F NMR
Therefore addition of D is to 3-re face
(bottom), which is called si with PEP addition
of ROH is to 2-si (top), which is 2-re in PEP
fluoromalate
fluoromalate
Anti addition
46
Stereochemistry of the Reaction Catalyzed by MurA
Not concerted
Scheme 6.39
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Friedel-Crafts reaction (alkylation)
Electrophilic Aromatic Substitution
Scheme 6.40
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Enzymatic Friedel-Crafts Reactions (alkylation in
nature)
Scheme 6.41
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Electrophilic Heteroaromatic Substitution porphobi
linogen deaminase
A acetate P propionate
porphyrins
corrins
heme
coenzyme B12
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Three Possible Mechanisms for the Reaction
Catalyzed by Porphobilinogen Deaminase
concerted
anionic
cationic
Scheme 6.43
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Substrate Analogues
substrate (but no tetrapyrrole formed-only
tripyrrole)
excellent substrates
not substrates
Consistent with E1 mechanism
Therefore E2? and E1cB unlikely
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Carbocation mechanism for porphobilinogen
deaminase
Cation Mechanism Most Reasonable
Scheme 6.44