The Organic Chemistry of Enzyme-Catalyzed Reactions: Revised Edition
The Organic Chemistry of Enzyme-Catalyzed Reactions: Revised Edition
The Organic Chemistry of Enzyme-Catalyzed Reactions: Revised Edition
Enzyme-Catalyzed Reactions
Revised Edition
Professor Richard B. Silverman
Department of Chemistry
Department of Biochemistry, Molecular
Biology, and Cell Biology
Northwestern University
Functional Parameters
Km Value
Ki Value
pI Value
Turnover Number
Specific Activity
pH Optimum
pH Range
Te mperature Optimum
Te mperature Range
Organism- related information
Organism
Source Tissue
Localization
Enzyme Structure
Sequence/ SwissProt link
3D-Structure/ PDB link
Molecular Weight
Subunits
Posttranslational Modification
Application & Engineering
Engineering
Application
Stability
pH Stability
Te mperature Stability
General Stability
Organic Solvent Stability
Oxidation Stability
Storage Stability
Disease & References
Disease
References
kon
k1
k1
koff
k1
Ks=
k1
Michaelis
complex
E .S
k2
E .P
E+P
Scheme 1.1
Turnover number
kcat (s-1)
10
102
103
103
103
103
106
106
107
kcat
Km
E S complex
Figure 1.1
Non-covalent interactions
electrostatic
(ionic)
O
+
RNH
ion dipo le
NH
R'
G = -RTlnKeq
If Keq = 0.01, G of -5.5 kcal/mol needed
to shift Keq to 100
NH
+
CH
COCH
CH
NMe
OH
Figure 1.2
Figure 1.3
Hydrophobic Interactions
When two nonpolar groups, each surrounded
by water molecules, approach each other, the
water molecules become disordered in an
attempt to associate with the water molecules
of the approaching group
Increases entropy, resulting in decrease in
the free energy (G = H-TS)
Binding Specificity
Can be absolute or can be very broad
Specificity of racemates may involve ES complex
formation with only one enantiomer or ES
complex formation with both enantiomers, but
only one is converted to product
Enzymes accomplish this because they are chiral
molecules (mammalian enzymes consist of only
L-amino acids)
EnzL+(R,S)
Scheme 1.2
EnzL
EnzL
diastereomers
Leu
H
H
OOC
Figure 1.4
OOC
NH
3
NH
3
Reaction Specificity
Unlike reactions in solution, enzymes can show
specificity for chemically identical protons
Figure 1.5
R'
R'
H
a
Rate Acceleration
An enzyme has numerous opportunities to
invoke catalysis:
Stabilization of the transition state
Destabilization of the ES complex
Destabilization of intermediates
Because of these opportunities, multiple
steps may be involved
Uncatalyzed
(G)
Uncatalyzed
FreeEnergy(G)
Catalyzed
EnzymeCatalyzed
E+S
ES
EP
E+P
ReactionCoordinate
Figure 1.6
ReactionCoordinate
Nonenzymatic rate
knon (s-1)
Enzymatic rate
kcat (s-1)
Rate acceleration
kcat/knon
cyclophilina
2.8 x 10-2
1.3 x 104
carbonic anhydrasea
1.3 x 10-1
106
50
chorismate mutasea
2.6 x 10-5
chymotrypsinb
4 x 10-9
4 x 10-2
triosephosphate
6 x 10-7
2 x 103
isomeraseb
fumaraseb
2 x 10-8
2 x 103
ketosteroid isomerasea
1.7 x 10-7
6.6 x 104
578
carboxypeptidase Aa
3 x 10-9
370
adenosine deaminasea
1.8 x 1010
ureaseb
3 x 10-10
3 x 104
alkaline phosphataseb
10-15
102
orotidine 5'-phosphate
39
2.8 x 10-16
decarboxylasea
a Taken from Radzicka, A.; Wolfenden, R. Science 1995, 267, 90.
b Taken from Horton, H.R.; Moran, L.A.; Ochs, R.S.; Rawn, J.D.; Scrimgeour,
K.G. Principles of Biochemistry; Neil Patterson: Englewood Cliffs, NJ, 1993.
4.6 x 105
7.7 x 106
1.9 x 106
107
3 x 109
1011
3.9 x 1011
1.9 x 1011
2.1 x 1012
1014
1017
1.4 x 1017
+CH3COO
Scheme 1.3
H3C
CH3
+ArO
)
rel
EffectiveMolarity(EM)
OAr
+CH
COO
3
OAr
220s
220
O
Decreasingrotationaland
translationalentropy
OAr
4
5.1x10
5.1x10
OAr
2.3x10
2.3x10
O
O
1.2x10
OAr
1.2x10
Covalent Catalysis
Nucleophilic catalysis
Activatedcarbonyl
X
R
Y
O
Y
O
anchimeric assistance
Most common
Cys (SH)
Ser (OH)
His (imidazole)
Lys (NH2)
Asp/Glu (COO-)
O
+
R
Z
1.1
Scheme 1.4
Cl
Cl
+
S
1.2
Scheme 1.5
HO
OH
18O
O Ar
18
CH3C O
H2O
(ArO)
Scheme 1.6
O
18O
18O
H2O
18O
18OH
OH
catalytic triad
Figure 1.7
H
N
NHR'
N
H
R1
Ser
R2
H
N
N
His
Scheme 1.7
OOC
Asp
pH7.9
pH7.9
pH7.3
pH7.3
[Buffer]
Figure 1.8
[Buffer]
Scheme 1.8
+ H2 O
poor
nucleophile
CH3COOH
EtOH
O
OC2H5
HO
strong
nucleophile
Scheme 1.9
H3C
O
OH
C2H5O
H3C
C2H5OH
O
H3C
OC2H5
+ H3
O+
H3C
OH
OC2H5
C
H3C + OC2H5
strong
electrophile
Scheme 1.10
H2O
H3C
OH
+ C2H5OH
O
R
B:
B+
Y
OH
base catalysis
Scheme 1.11
acid catalysis
Hb+
pKa=3.4
HO Ha
HO
Ha
OHb
Hc+
Ph
pKa~8
HO
1.4
1.3
pKE=18.6
Ph
Ha+
OHc
OHb
Ha
pKE=15.4
pKa=22.0
Ph
HO
O
OHb
Hc+
Ph
OHc
Ha+
Ph
pKa=6.6
HO
OHb
pKa~7.4
HO
1.6
Scheme 1.12
OHc
+
Ha
1.5
OHb
O
A
R
H
weak
base
R=H,alkyl,SR'
strong
base
X
O
One-base
mechanism
syn-elimination
stronger acid
needed
H
strong
acid
B+
low-barrier
H-bond
:B
B:
BH
B
HX
M+
B:
M+
B
H
O
O
Two-base
mechanism
anti-elimination
M+
weak acid
B:
carboxylic acids
Scheme 1.13
O
R
B:
Scheme 1.14
O
R
H
B+
O
R'
O
R
B:
Scheme 1.15
R'
R
H
H
B+
Electrostatic Catalysis
Electrostatic stabilization of the transition state
oxyanion hole
alsocouldbea
Hbondordipole
+
+
O
H
N
R
R'
N
H
H
N
O
Scheme 1.16
R"
N
H
H
N
R
N
O H
R'
H
N
O
R"
N
H
Desolvation
The removal of water molecules at the active site
on substrate binding
Exposes substrate to lower dielectric
constant environment
Exposes water-bonded charged groups for
electrostatic catalysis
Destabilizes the ground state
Strain Energy
Alkaline hydrolysis of phosphodiesters
O
O
OH
1.7
CH3 CH3
HO
O
P
O
O
P
1.8
Scheme 1.17
k1.7
k1.8
= 108
OH
CH3
CH3
HO
O
O
O
P
O
Figure 1.9
Figure 1.10
COO
Lys252
O
NH2
COO
: NH2
Lys252
Lys252
NH
O
ZnB(Cys)4
NH2
strainenergy
electrostaticcatalysis
approximation
COO
COO
COO
NH
:B
ZnB(Cys)4
NH2
basecatalysis
(X3)ZnA
ZnB(Cys)4
COO
Lys252
NH2
NH
..
H2N
(X3)ZnA
NH2
COO
B:
Lys252
COO
HO
:B
approximation
COO
basecatalysis
NH
O
OH
COO
+
NHLys252
+
NH
(X)3ZnA
+
NHLys252
..
NH
HO
NH2
COO
(X)3ZnA
NH2
basecatalysis
strainenergy
electrostaticcatalysis
COO
COO
COO
COO
H
HO
(X)3ZnA
NH2
N
H
HO
(X)3ZnA
NH2
N+
H
basecatalysis
B:
:B
H
basecatalysis
HO
(X)3ZnA
COO
NHLys252
NH2
COO
N+
H
acidcatalysis