The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 6
Substitutions
SN1
Reactions catalyzed by farnesyl diphosphate
synthase
-PPi
+
PPO
6.1
isopentenyl DP
PPO
geranyl
diphosphate
PPO
6.2
6.3
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
CHF2
CH2F
PPO
PPO
6.8
Relative
rate
1.75  10-2
CF3
PPO
6.9
6.10
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)
Carbocation Mechanism (SN1) for
Farnesyl Diphosphate Synthase
+
PPO
R
R
6.11
R = Me (6.2)
R = C5H11 (6.3)
+
PPO
+ PPi
R
+
PPO
H
B:
Scheme 6.2
R
PPO
R
Stereochemistry of Farnesyl Diphosphate
Synthase
syn addition/elimination
PPO
6.21
si
PPO
B:
HR
HS
6.20
Figure 6.1
Sesquiterpenes Biosynthesized from Farnesyl DP
Reaction catalyzed by pentalenene synthase
309His
309His
N
NH
NH
N
H
H
6.22
PPO
humulene
309His
Mg2+
N
309His
NH
N
H
H
H
H
NH
H
H
6.24
pentalenene
6.23
Scheme 6.5
From the Crystal Structure of Pentalenene Synthase
Stabilization of carbocation intermediates by
active-site phenylalanine and asparagine residues
cation- interaction


O
NH2
C
219Asn
Phe77
Figure 6.2
carbocation
stabilization
SN1/SN2
Reaction catalyzed by phosphorylases
O
R O
R'
+
-O
P OOH
Scheme 6.6
O
RO P
O-
O-
+
R'OH
Reaction Catalyzed by Disaccharide
Phosphorylases
OH
OH
another sugar
O
OH
+
HO
18O
HO
O
OH
Pi
OPO3= +
HO
R
6.25
Scheme 6.7
HO
6.26
H18OR
6.27
Stereochemistry of the Reactions Catalyzed by
Disaccharide Phosphorylases
cellobiose phosphorylase
maltose phosphorylase
sucrose phosphorylase
C-1 Configuration
of Disaccharide



C-1 Configuration
of Phosphorylated Product



inversion
retention
Two Mechanisms for Inversion
SN2 versus stereospecific SN1 reaction
OH
O
SN2
OH
SN1 S 2
N
OH
HO
:
O
HO
-OPO H3
OH
OPO3H-
OH
HO
HO
OR
Scheme 6.8
+
O
SN1
-OPO H3
OH
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
HO
HO
HO
O
HO
O
OH
HO
glucosyl
fructosyl
6.28
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
Experiments to Identify Active Site Residue (X)
1. [14C] glucosyl enzyme
MeOH
6.29 (R = H), not 6.29 (R = Me)
(glucose)
OH
O
OH
O
X
HO
HO
OR
HO
OH
HO
OH
6.29
2. [14C] glucosyl enzyme very sensitive to base
3. [14C] glucosyl enzyme
NH2OH
O
6.29 (R = H) +
NHOH
Therefore X is Glu or Asp
Disaccharide Phosphorylase Reactions
Involving an Active-site Carboxylate
OH
OH
HO
O
.. O- SN2
O
HO
B+
O
SN2
HO
-OPO
3H
-
HO
6.31
H
SN2
a
SN1
OH
+
OH
Scheme 6.9
O
OH
OR
HO
O
OH
O
a
-
OH
O
O
b
-OPO
HO
6.32
b
OH
3H
O
-
HO
HO
OPO3H-
Two Mechanisms for Reactions Catalyzed by
-Glycosidases--Hydrolysis of Disaccharides
acid
OH
O
HO
HO
A
Scheme 6.10
OH
O
O
H O
O
-ROH
R
HO H OH
HO
HO
HO
O
OH
HO
-O
SN2 (inversion)
O
(General acid/base mechanism)
O
O
base
acid
B
H O
OH
O
HO
HO
HO
OR
-O
O
-ROH
HO
HO
OH
O
OH
HO O
O
nucleophile
Two active site
carboxylic acids
H O
O
HO
HO
OH HO
O OH
HO
O
-O
O
double
SN2
O (retention)
(Covalent)
Mutation to Ala: kcat 107-fold lower
Add in N3- to replace the carboxylate nucleophile:
kcat only 102-fold lower (-azide forms)
Differentiation of SN2 from SN1
for -Glycosidases
OH
O
X = F or
X
O
HO
HO
F
really good
leaving groups
NO2
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
HO
HO
OH
O
OH
O
+
F
O
O
HO
HO
OH
O
OH
HO
HO
6.35
6.34
after isolation
(Glu-358)
Ph
HO
HO
O HO
Ph
OH
F
6.36
OH
O
OH
O
X
Scheme 6.11
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
HO
HO
OH
O
H
OR
B
SN2-like
OH
O
HO
HO
HO
O–
substitution of
Glu-358 by Asn
or Gln - inactive
by Asp - 2500x
slower
OH
O
O
‡
B–
OH
OR
HO
HO
O
H
HO
O–
O
O H
O
SN1-like
H B
HO
HO
OH
O
OH
O
Scheme 6.12
H
OH
O–
B
HO
HO
OH OH
O+
HO O–
O
‡
SN2
Two mechanisms for epoxide hydrolase
General base mechanism
A
OH
O
Enz B:
H
EnzBH
OH
+
HO
Nucleophilic (covalent) mechanism
O
B
Enz
B
O
O–
Enz
O
OH
O
O
Enz
OH
O–
+
HO
BH+
B:
H OH
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
Further Evidence for Ester Linkage
Covalent intermediate isolated during reaction
catalyzed by epoxide hydrolase
H+
O 3
H
OH 3H
CO2Me
O
O
O-
OH 3H
CO2Me
O
6.39
6.38
Asp333
N
H OH Asp
quenched (AcOH)
333
precipitated (acidic acetone)
NH
OH 3H
OH 3H
OH
HO
LiAlH4
O
6.40
Scheme 6.15
CO2Me
HO
CO2Me
O
Asp333
isolated
NaOH
OH 3H
CO2H
HO
6.41
A Catalytic Antibody-catalyzed 6-Endo-tet
Ring Closure
Baldwin’s rules predict 5-exo-tet
a
HO
6-endo-tet
O
Ar
6.43
O
b
b
Ar
obtained with a
catalytic antibody
(anti-Baldwin product)
Scheme 6.16
a
HO
5-exo-tet
H
a
:O
b
H
6.42
O
Ar
H
1.8 kcal/mol lower in
energy in solution
SN2
Reaction catalyzed by isochorismate synthase
COO-
COO18OH
O
COO-
OH
6.44
Scheme 6.18
isochorismate
synthase
Mg++ H218O
O
6.45
COO-
SN2 Mechanism for Isochorismate Synthase
H
COO-
18O
:B
-OOC
‡
O
H
COO18OH
COOO
COO-
OH
B+ H
6.44
O
H
H
O
2+
Mg
O18
H
6.46
all axial conformation
Scheme 6.19
COO-
Reaction Catalyzed by Anthranilate Synthase
CO2-
CO2NH3
O
CO2-
CO2-
-H2O
O
OH
6.44
Scheme 6.20
NH3+
NH3
CO2-
6.47
synthesized kinetically competent
intermediate
6.48
Reaction Catalyzed by p-Aminobenzoic Acid (PABA)
Synthase
synthesized kinetically competent
Scheme 6.21
CO2-
CO2-
CO2-
NH3
O
OH
6.44
CO2-
-H2O
O
CO2-
+NH
3
6.49
reaction different from others
NH3+
6.50
Synthesized as TS‡ Mimics of the 3 Enzymes
(in the all-axial conformation)
isochorismate synthase
CO2-
anthranilate synthase PABA synthase
CO2-
OH
O
OH
6.51
CO2-
O
OH
6.52
CO2-
+
NH3
OH
CO2-
O
+ NH
CO2-
3
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-
H
N
+
H3N
O
O
N
H
COO-
SH
6.57
g-glutamylcysteinylglycine
SNAr
Reaction catalyzed by glutathione S-transferase
Tyr
O
Tyr
GS
Scheme 6.23
O
H
X
O
N O+
slow
H
_
O
X
_
N O
+
GS
GS
NO2
fast
+ HX
Y
Y
Hammett study
 = +1.2 for GSH
= +2.5 for g-Glu-Cys
rate X = F > X = Cl
Y
therefore carbanionic
Glutathione S-transferase-Catalyzed Reaction
of Glutathione with 1,3,5-Trinitrobenzene
O2N
NO2
N+
O
O-
GS
GS-
H
NO2
O2N
NO26.58
Scheme 6.24
observed spectroscopically
Meisenheimer complex
Electrophilic Substitution
(Addition/Elimination Mechanism)
Reaction catalyzed by 5-enolpyruvylshikimate3-phosphate (EPSP) synthase
CO2-
CO2+
=O PO
3
OH
OH
+ Pi
=O PO
3
CO26.61
6.60
shikimate-3-P
=O PO
3
O
CO2-
OH
6.62
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
:
-O C
2
CH2
addition
+
OPO3=
RO
H
- Pi
OPO3=
CO2-
RO
elimination
6.62
CO2-
B:
2) Stepwise
H B+
:
-O C
2
OPO3=
ROH
+
-O C
2
+
RO
OPO3=
H
B:
OPO3=
CO2-
B:
Scheme 6.30 (continued on next slide)
H
RO
CH2
+
CO2-
6.62
4 Possible Mechanisms for EPSP Synthase (continued)
3) Covalent-concerted (a) and
4) Covalent-stepwise (b)
X-O
2C
H B+
:B
H
H2C
b
b
X
OPO3=
stepwise
_
CO2
a
H B+
OPO3=
RO H
a
B:
X
concerted
H
H2C
X
CH3
:B
_
X
CO2
Scheme 6.30
ROH
_
CO2
6.62
+
_
CO2
:
RO
RO
_
CO2
H2C
X
:B
H
_
CO2
RO
H
:B
Isolated by Et3N Quench
CO2CH3
=O PO
3
O
OH
OPO3=
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-
F
CO2F
CH2F
=O PO
3
OH
=O PO
3
OH
6.60
COO-
6.65
=O PO
3
O
OH
6.66
CO2OPO3=
+ Pi
=O PO
3
O
CO2-
OH
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-
CO2CH3
=O PO
3
CO2-
O
OH
OPO3=
6.68
Scheme 6.32
CH3
=O PO
3
O
OH
6.69
CO2-
EPSP
To Determine Stereochemistry of Tetrahedral
Intermediate
phosphonate (stable)
CO2-
CO2CH3
=O PO
3
O
OH
PO3=
CO2-
CH3
=O PO
3
O
OH
CO2PO3=
6.71
6.72
Ki = 15 nM*
(suggests this
stereochemistry)
Ki = 1130 nM
To make a stable phosphate, put in an electron
withdrawing group
CH2F, CHF2, CF3
CO2-
CO2X
=O
3PO
O
OH
6.73
OPO3=
CO2-
X
=O
3PO
O
OH
CO2OPO3=
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-Nacetylglucosamine enolpyruvyl transferase (MurA)
OH
O
O
HO
HO
O
NH
O
OH
NH
O
O
P O P O
OO6.75
N
O
+
O
HO
OH
=O
3PO
CO2-
-Pi
HO
O
COO-
O
O
O
NH
O
NH
O
O
P O P O
OO6.76
N
O
O
HO
OH
Scheme 6.34
opposite results
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
covalent
intermediate
B
H
OPO3
OPO3=
CO2-
=
CO2-
UDP-GlcNAc
X
X-
6.77
phospholactyl enzyme
kinetically competent
Scheme 6.35
noncovalent
intermediate
HO
O
OH
O
- HN O-UDP
CO2
O
OPO3=
6.78
OH
HO
O
CO2-
O
+ Pi
HN O-UDP
O
6.79
Further Evidence for Covalent and
Noncovalent Intermediates
Inactivation of MurA by (E)- and (Z)-3-fluoroPEP
OPO3=
H
F
OPO3=
H
CO2-
or
CO2-
F
6.80
Scheme 6.36
OPO3=
CO2FCH2
X
6.65
-O C
2
OH
O
HO
O
OPO3=
FCH2
NH
O UDP
Ac
6.81
6.82
covalent
(stable)
noncovalent
Kinetics suggest that 6.82 does not come from 6.81
Branching Mechanism
More consistent mechanism for the reaction
catalyzed by MurA
+O—PO =
3
OPO3=
H
H
CO2H
B
H3C
CO26.83
RO:
noncovalent
intermediate
OPO3=
X
CO2-
H3C
- Pi
6.84
X-
CH3
ROH
OPO3=
CO2-
6.86
covalent
intermediate
CO2-
H
H
H
O+
R
B-
H
CO2-
H
OR
6.79
6.85
Scheme 6.37
Determination of
the Stereochemistry
of the Reaction
Catalyzed by MurA
Scheme 6.38
H
OPO3=
H
From crystal
structure [2R];
therefore ROH
addition is 2-si
(top) (2-re in
PEP)
OR
CO2-
F
OPO3=
H
CO2-
6.80
6.65
MurA
ROH/D2O
OR
H
CO2D
F OPO3
6.87E
H
F
D
H
[2R]
OPO3
[2R]
-UDP-GlcNAc
O
F
CO2-
D
OR
CO2-
6.87Z
alkaline
phosphatase
-UDP-GlcNAc
F
OPO3=
CO2D
3R
2R
6.87E
F
O
CO2-
D
F 6.88E
fluoropyruvate
H 6.88Z
pyruvate
carboxylase
fluorooxaloacetate
-OOC
Analyzed for H or D by 19F NMR
(retention)
O
-OOC
CO2-
D
F
O
CO2-
H
F
malate
Therefore addition of D+ is to 3-re
dehydrogenase
face (bottom), which is called si
OH
with PEP; addition of ROH is to fluoromalate
-OOC
-OOC
H
CO22-si (top), which is 2-re in PEP
H
D
F
F
Anti addition
6.89E
OH
H
fluoromalate
CO26.89Z
Stereochemistry of the Reaction
Catalyzed by MurA
HO
O
H
B:
OH
O
re
re
NH
Ac O UDP
H
OPO3=
H
COO-
si
anti
addition
HO
O
H
OPO3=
H
H
H
S
115Cys
OH
O
COO-
NH
Ac
O UDP
syn
elimination
COO-
H
S
H
115Cys
S
115Cys
Not concerted
Scheme 6.39
HO
O
H
HPO4=
OH
O
NH
O UDP
Ac
si
Electrophilic Aromatic Substitution
Friedel-Crafts reaction (alkylation)
R' H
R' X
AlCl3
R
Scheme 6.40
-X
R'
+
R
R
Enzymatic Friedel-Crafts Reactions
(alkylation in nature)
COO-
COOCH3
+
O
OPP
CH3
H
8
H
8
O
B+
H
CH3
:B
B:
COOcoenzyme Q
vitamin K
other quinones
8
OH
Scheme 6.41
CH3
Electrophilic Heteroaromatic Substitution
porphobilinogen deaminase
COOH
HOOC
H2N
A = acetate
P = propionate
N
H
6.90
P
A
HO
N
H
N
H
N
H
P
P A
P A
A
N
H
6.91
porphyrins
heme
corrins
coenzyme B12
1) E2' (1,6-elimination)
Three Possible Mechanisms
for the Reaction Catalyzed by
Porphobilinogen Deaminase
concerted
P
A
P
A
N
NH3+
Nu
N
H
N
Nu
H
B
2) E1cB
P
A
anionic
P
A
Nu
N
NH3+
N
NH3+
H
B
3) E1
cationic
Scheme 6.43
P
A
NH3+
N
H
P
A
P
A
P
A
N
N
H
H
Substrate Analogues
P
A
N
NH2
CH3
6.92
P
A
P
A
F3C
H3C
NH2
N
H
6.93
substrate
(but no tetrapyrrole
formed-only tripyrrole)
Therefore E2 and
E1cB unlikely
F3C
H3C
NH2
N
H
6.94
excellent
substrates
P
A
P
A
OH
N
H
OH
6.95
N
H
6.96
not substrates
Consistent with E1 mechanism
Cation Mechanism Most Reasonable
Carbocation mechanism for
porphobilinogen deaminase
A
A
A
P
NH2
N
NH2 H
N
H
P
N
H
-NH3
P
A
A
P
P
A
+N
H H
N+
H
NH2
+ H
B
P
A
N
H
P
N
H
NH2
B:
6.91
HO
P
N
H
P
A
Scheme 6.44
A
N+
H
H
:B
P
P A
A
N
H
N
H
P
A
N
H