The Organic Chemistry of
Enzyme-Catalyzed Reactions
Revised Edition
Professor Richard B. Silverman
Department of Chemistry
Department of Biochemistry, Molecular
Biology, and Cell Biology
Northwestern University
The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 1
Enzymes as Catalysts
For published data regarding any enzyme see:
http://www.brenda-enzymes.info/
Nom e ncla tu re
En z ym e Na me s
EC Nu mb er
Com m on / Re com m en d e d Na m e
S yste ma tic Na me
S yno n ym s
CAS Re gi str y N um be r
Funct io nal P ar am e te rs
Km V al ue
Ki Valu e
pI Val ue
T u rn o ver Nu mb er
S p e cific Activity
pH Op tim u m
pH Ra n g e
Te mp er a tu re Op tim u m
Te mp er a tu re R a n g e
Org an is m - rel ated info rm at io n
Orga n is m
S o ur ce T is s ue
Lo ca liza tion
Enz ym e St ruc tu re
Se qu e n ce / Swis sP rot lin k
3 D-Str uc tur e / PDB lin k
Mo lec u la r Weig h t
S ub u n it s
Po s ttr an s la tio n al Modif ica tion
Ap plicat io n & Eng in e er in g
En g in eer in g
Ap p lica tio n
Rea cti o n & Spec ificity
Path wa y
Cat alys e d Re ac tio n
Re ac tion Ty p e
Na tu ra l S ub s t ra tes and P ro d u cts
S ub s t ra t es and P ro d u cts
S ub s t ra t es
Na tu ra l S ub s t ra te
Pro duc ts
Na tu ra l Pro duc t
In h ibi to rs
Co facto rs
Met a ls / Io n s
Act iva tin g Co mp ou nd s
Lig and s
Is o lati o n & Prep aratio n
Pu rifica tio n
Clon e d
Re na tu re d
Crys t alliza tio n
St abi lity
pH St abi lity
Te mp er a tu re St abi lity
Ge n er a l St ab ilit y
Orga n ic So lve nt St abil ity
Ox ida tion S t abil ity
S t or a ge Stab ilit y
Dise as e & Re fe re nc e s
Dis e a se
Re fe ren ces
What are enzymes, and how do they work?
• First “isolation” of an enzyme in 1833
• Ethanol added to aqueous extract of malt
• Yielded heat-labile precipitate that was
utilized to hydrolyze starch to soluble sugar;
precipitate now known as amylase
• 1878 - Kühne coined term enzyme - means
“in yeast”
• 1898 - Duclaux proposed all enzymes should
have suffix “ase”
• Enzymes - natural proteins that catalyze
chemical reactions
• First enzyme recognized as protein was jack
bean urease
• Crystallized in 1926
• Took 70 more years (1995), though, to obtain
its crystal structure
• Enzymes have molecular weights of several
thousand to several million, yet catalyze
transformations on molecules as small as
carbon dioxide and nitrogen
• Function by lowering transition-state energies
and energetic intermediates and by raising
the ground-state energy
• Many different hypotheses proposed for how
enzymes catalyze reactions
• Common link of hypotheses: enzymecatalyzed reaction always initiated by the
formation of an enzyme-substrate (or ES)
complex in a small cavity called the active site
• 1894 - Lock-and-key hypothesis - Fischer
proposed enzyme is the lock into which the
substrate (the key) fits
• Does not rationalize certain observed
phenomena:
Compounds having less bulky substituents often
fail to be substrates
Some compounds with more bulky substituents
bind more tightly
Some enzymes that catalyze reactions between
two substrates do not bind one substrate until
the other one is bound
1958 - Induced-fit hypothesis proposed by
Koshland:
When a substrate begins to bind to an enzyme,
interactions induce a conformational change
in the enzyme
Results in a change of the enzyme from a low
catalytic form to a high catalytic form
Induced-fit hypothesis requires a flexible active
site
Concept of flexible active site stated earlier by
Pauling (1946):
Hypothesized that an enzyme is a flexible
template that is most complementary to
substrates at the transition state rather than
at the ground state
Therefore, the substrate does not bind most
effectively in the ES complex
As reaction proceeds, enzyme conforms better
to the transition-state structure
Transition-state stabilization results in rate
enhancement
• Only a dozen or so amino acid residues may
make up the active site
• Only two or three may be involved directly in
substrate binding and/or catalysis
Why is it necessary for enzymes to be so large?
• Most effective binding of substrate results
from close packing of atoms within protein
• Remainder of enzyme outside active site is
required to maintain integrity of the active site
• May serve to channel the substrate into the
active site
Active site aligns the orbitals of substrates and
catalytic groups on the enzyme optimally for
conversion to the transition-state structure-called orbital steering
• Enzyme catalysis characterized by two
features: specificity and rate acceleration
• Active site contains amino acid residues and
cofactors that are responsible for the above
features
• Cofactor, also called a coenzyme, is an
organic molecule or metal ion that is essential
for the catalytic action
Specificity of Enzyme-Catalyzed Reactions
• Two types of specificity: (1) Specificity of binding
and (2) specificity of reaction
Specificity of Binding
• Enzyme catalysis is initiated by interaction
between enzyme and substrate (ES complex)
• k1, also referred to as kon, is rate constant for
formation of the ES complex
• k-1, also referred to as koff, is rate constant for
breakdown of the complex
• Stability of ES complex is related to affinity of
the substrate for the enzyme as measured by Ks,
dissociation constant for the ES complex
Generalized enzyme-catalyzed reaction
kon
k1
Michaelis
complex
E .S
E + S
k-1
koff
Ks =
k-1
k2
E .P
E + P
Scheme 1.1
k1
When k2 << k-1,
k2 called kcat (turnover number)
Ks called Km (Michaelis-Menten constant)
kcat represents the maximum number of substrate
molecules converted to product molecules per
active site per unit of time; called turnover number
Table 1.1. Examples of Turnover Numbersa
Enzyme
papain
carboxypeptidase
acetylch olinesterase
kinases
dehydrogenases
aminotransferases
carbonic anhydrase
superoxide dismuta se
catalase
aEigen,
Turnover number
kcat (s-1)
10
102
103
103
103
103
106
106
107
M.; Hammes, G.G. Adv. Enzymol. 1963, 25, 1.
• Km is the concentration of substrate that
produces half the maximum rate
• Km is a dissociation constant, so the smaller
the Km the stronger the interaction between E
and S
• kcat/Km is the specificity constant - used to
rank an enzyme according to how good it is
with different substrates
Upper limit for
kcat
Km
is rate of diffusion (109 M-1s-1)
How does an enzyme release product so
efficiently given that the enzyme binds the
transition state structure about 1012 times more
tightly than it binds the substrate or products?
After bond breaking (or making) at transition
state, interactions that match in the transitionstate stabilizing complex are no longer present.
Therefore products are poorly bound, resulting in
expulsion.
As bonds are broken/made, changes in electronic
distribution can occur, generating a repulsive
interaction, leading to expulsion of products
E • S complex
Figure 1.1
Non-covalent interactions
electrostatic
(ionic)
+
RNH3
R
ion-dipole
O
O C

O
 C
+
NH3
R'
R
dipole-dipole

 O
H
C
O
R'
O
H-bonding
RC
charge
transfer
D
A
O
hydrophobic
RC O
O
H
O
H
A
D

∆Gº = -RTlnKeq
If Keq = 0.01, ∆Gº of -5.5 kcal/mol needed
to shift Keq to 100
Specific Forces Involved in
E•S Complex Formation
Examples of ionic, ion-dipole, and dipole-dipole
interactions. The wavy line represents the
enzyme active site
ion-dipole
ionic
+


NH3 O
O
+ O
CH3COCH2CH2NMe3

OH
Figure 1.2

dipole-dipole
Hydrogen bonding in the secondary structure of
proteins: -helix and -sheet.
H-bonds
H-bonds
A type of dipole-dipole
interaction between X-H
and Y: (N, O)
Figure 1.3
Charge Transfer Complexes
• When a molecule (or group) that is a good
electron donor comes into contact with a
molecule (or group) that is a good electron
acceptor, donor may transfer some of its
charge to the acceptor
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-TS)
van der Waals Forces
• Atoms have a temporary nonsymmetrical
distribution of electron density resulting in
generation of a temporary dipole
• Temporary dipoles of one molecule induce
opposite dipoles in the approaching molecule
Binding Specificity
• Can be absolute or can be very broad
• Specificity of racemates may involve E•S complex
formation with only one enantiomer or E•S
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)
Binding specificity of enantiomers
Resolution of a racemic mixture
EnzL + (R,S)
EnzL
R
+
EnzL
diastereomers
Scheme 1.2
S
• Binding energy for E•S complex formation
with one enantiomer may be much higher
than that with the other enantiomer
• Both E•S complexes may form, but only one
E•S complex may lead to product formation
• Enantiomer that does not turn over is said to
undergo nonproductive binding
Steric hindrance to binding of enantiomers
Basis for enantioselectivity in enzymes
A
B
Leu
H
OOC
Figure 1.4
H
S
NH3
OOC
R
NH3
Reaction Specificity
Unlike reactions in solution, enzymes can show
specificity for chemically identical protons
Enzyme specificity for chemically identical
protons. R and R on the enzyme are
groups that interact specifically with R and
R, respectively, on the substrate.
enzyme
Figure 1.5
R
R'
R
R'
B- Ha Hb
Rate Acceleration
• An enzyme has numerous opportunities to
invoke catalysis:
– Stabilization of the transition state
– Destabilization of the E•S complex
– Destabilization of intermediates
• Because of these opportunities, multiple
steps may be involved
A
Free Energy (²G)
Effect of (A) a chemical catalyst and
(B) an enzyme on activation energy
B
Uncatalyzed
Uncatalyzed
Catalyzed
Enzyme Catalyzed
E+S
ES
EP
E+P
Reaction Coordinate
Figure 1.6
Reaction Coordinate
1010-1014 fold typically
Enzyme catalysis does not alter the equilibrium
of a reversible reaction; it accelerates attainment
of the equilibrium
Table 1.2. Examples of Enzymatic Rate Acceleration
Enzyme
Nonenzymatic rate
knon (s-1)
Enzymatic rate
kcat (s-1)
Rate acceleration
kcat/knon
cycl ophilina
2.8 x 10-2
1.3 x 104
carbonic anhydrasea
1.3 x 10-1
106
50
chorismate muta sea
2.6 x 10-5
chymotrypsinb
4 x 1 0-9
4 x 1 0-2
triosephosphate
6 x 1 0-7
2 x 1 03
isomeraseb
fumaraseb
2 x 1 0-8
2 x 1 03
ketosteroid isomerasea
1.7 x 10-7
6.6 x 104
578
carboxypeptidase Aa
3 x 1 0-9
370
adenosine deaminasea
1.8 x 1010
ureaseb
3 x 1 0-10
3 x 1 04
alkaline phosphataseb
10-15
102
orotidine 5'-phosphate
39
2.8 x 10-16
decarboxyl asea
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 1 09
1011
3.9 x 1011
1.9 x 1011
2.1 x 1012
1014
1017
1.4 x 1017
Mechanisms of Enzyme Catalysis
Approximation
• Rate enhancement by proximity
• Enzyme serves as a template to bind the
substrates
• Reaction of enzyme-bound substrates
becomes first order
• Equivalent to increasing the concentration of
the reacting groups
• Exemplified with nonenzymatic model studies
Second-order reaction of acetate with
aryl acetate
O
CH3COAr
+ CH3COO-
Scheme 1.3
H3C
O
O
C
C
O
CH3
+ ArO-
Table 1.3. Effect of Approximation on Reaction Rates
Relative rate (krel )
Effective Molarity (EM)
O
1 M-1 s-1
OAr
+ CH 3 COOO
OAr
O
220 s-1
220 M
5.1 x 10 4 s-1
5.1 x 10 4 M
2.3 x 10 6 s-1
2.3 x 10 6 M
1.2 x 10 7 s-1
1.2 x 10 7 M
-
O
Decreasing rotational and
translational entropy
O
OAr
OO
O
OAr
O
-
O
O
O
OO
OAr
Covalent Catalysis
Nucleophilic catalysis
Activated carbonyl
X
X
R
Y
O
R
X
-Y-
O
+
Y
R
O-
anchimeric assistance
Most common
X-
Cys (SH)
Ser (OH)
His (imidazole)
Lys (NH2)
Asp/Glu (COO-)
Z-
O
X
R
1.1
Scheme 1.4
Z
Anchimeric assistance by a neighboring group
-Cl-
S
+
S
Cl
S
OH
1.2
Scheme 1.5
HO-
Model Reaction for Covalent Catalysis
Early evidence to support covalent catalysis
O
18O
O Ar
CH3C
O
18
O-
H2O
(-ArO-)
Scheme 1.6
O
18O
18O
18O
H2O
18OH
+
OH
General Acid/Base Catalysis
This is important for any reaction in which proton
transfer occurs
The catalytic triad of -chymotrypsin. The
distances are as follows: d1 = 2.82 Å; d2 =
2.61 Å; d3 = 2.76 Å.
catalytic triad
Figure 1.7
Charge relay system for activation of an activesite serine residue in -chymotrypsin
R
O
H
N
NHR'
N
H
R1
Ser
R2
O
O
H
N
N
His
Scheme 1.7
H
-OOC
Asp
• pKa values of amino acid side-chain groups within
the active site of enzymes can be quite different
from those in solution
• Partly result of low polarity inside of proteins
Molecular dynamics simulations show
interiors of these proteins have dielectric
constants of about 2-3 (dielectric constant for
benzene or dioxane)
• If a carboxylic acid is in a nonpolar region, pKa will
rise
• Glutamate-35 in the lysozyme-glycolchitin complex
has a pKa of 8.2; pKa in solution is 4.5
• If the carboxylate ion forms salt bridge, it is
stabilized and has a lower pKa
• Basic group in a nonpolar environment has a
lower pKa
• pKa of a base will fall if adjacent to other
bases
• Active-site lysine in acetoacetate
decarboxylase has a pKa of 5.9 (pKa in
solution is 10.5)
Two kinds of acid/base catalysis:
• Specific acid or specific base catalysis catalysis by a hydronium (H3O+) or hydroxide
(HO-) ion, and is determined only by the pH
• General acid/base catalysis - reaction rate
increases with increasing buffer concentration
at a constant pH and ionic strength
Effect of the buffer concentration on (A)
specific acid/base catalysis and (B)
general acid/base catalysis
B
A
k
pH 7.9
pH 7.9
k
pH 7.3
pH 7.3
[Buffer]
Specific acid/base catalysis
Figure 1.8
[Buffer]
General acid/base catalysis
Specific Acid-Base Catalysis
Hydrolysis of ethyl acetate
O
H3C C OEt
weak
electrophile
Scheme 1.8
+ H2O
poor
nucleophile
CH3COOH
+
EtOH
Alkaline hydrolysis of ethyl acetate
O
H3C
C
O
OC2H5
HOstrong
nucleophile
Scheme 1.9
H3C
C
O
OH
+
C2H5O-
H3C
C
O-
+
C2H5OH
Acid hydrolysis of ethyl acetate
+OH
O
H3C
C
OC2H5
+ H3
O+
H3C
C
OH
OC2H5
C
H3C + OC2H5
strong
electrophile
Scheme 1.10
O
H2O
H3C
C
OH
+ C2H5OH
Enzymes can utilize acid and base
catalysis simultaneously
Simultaneous acid and base enzyme catalysis
B+
O
Y
R
B:
H
H
OH
base catalysis
Scheme 1.11
acid catalysis
Simultaneous acid/base catalysis is the reason for
how enzymes are capable of deprotonating weak
carbon acids
Simultaneous acid and base enzyme catalysis
in the enolization of mandelic acid
O
Ph
O-
± Hb+
pKa = 3.4
HO Ha
O
Ph
HO
Ha
+
OHb
± Hc+
Ph
pKa ~ -8
HO
OHc
OHb
Ha
1.4
1.3
pKE = 18.6
± Ha+
pKE = 15.4
pKa = 22.0
Ph
HO
OOHb
Hc+
Ph
OHc
± Ha+
Ph
pKa = 6.6
HO
OHb
pKa ~ 7.4
HO
±
1.6
Scheme 1.12
OHc
+
Ha
1.5
OHb
• Low-barrier hydrogen bonds - short (< 2.5Å),
very strong hydrogen bonds
• Stabilization of the enolic intermediate occurs
via low-barrier hydrogen bonds
Simultaneous acid and base enzyme catalysis in the
1,4-elimination of -substituted (A) aldehydes,
ketones, thioesters and (B) carboxylic acids
low-barrier
H-bond
H
B
:B
H
O
A
O
X
R
H
“weak”
base
O
X
R
R
“strong”
acid
H
H
B:
One-base
mechanism
syn-elimination
B+
R = H, alkyl, SR'
low-barrier
H-bond
B
H
H
X
“strong”
base
B:
X
O–
H
B
H
B
O
B
stronger acid
needed
O
BH
B
-HX
O–
M+
H
H
M+
B
H
O
O
Two-base
mechanism
anti-elimination
M+
“weak” acid
B:
Scheme 1.13
carboxylic acids
ElcB mechanism - not relevant
Base catalyzed 1,4-elimination of -substituted
carbonyl compounds via an enolate
intermediate (ElcB mechanism)
Needs acid or metal catalysis
O
X
O
R
H
H
B:
Scheme 1.14
X
O
R
H
B+
R
Alternative to Low-Barrier Hydrogen Bond
Electrostatic enzyme catalysis in enolization
O
R'
O
R
H
H
B:
Scheme 1.15
R'
R
H
H
B+
Electrostatic Catalysis
Electrostatic stabilization of the transition state
oxyanion hole
also could be a
H bond or dipole
+
+
O
H
N
R
R'
N
H
O
H
N
O
O
Scheme 1.16
R"
N
H
O
H
N
R
N
O H
R'
O
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
P
-O
O-
CH3 CH3
HO
-O
O
P
O-
O
O
P
O
-O
1.7
O1.8
Scheme 1.17
k1.7
k1.8
= 108
CH3
-OH
CH3
HO
-O
-O
O
P
O
Induced Fit Hypothesis
putting strain energy into the substrate
Figure 1.9
Energetic Effect of Enzyme Catalysis
Figure 1.10
Importance of ground state destabilization
Mechanisms of Enzyme Catalysis - porphobilinogen synthase
approximation
covalent catalysis
COO-
Lys252
O
NH2
COO-
COOLys252
: NH2
Lys252
NH
O
ZnB(Cys)4
NH2
strain energy
electrostatic catalysis
H
NH
:B
NH2
ZnB(Cys)4
OH
ZnB(Cys)4
base catalysis
approximation
COOCOO-
COOLys252
COO-
Lys252
COO-
B:
H
NH
(X3)ZnA
N
O
..
H2N
NH2
(X3)ZnA
NH2
H
H
HO
:B
(X)3ZnA
+
NH-Lys252
..
NH
HO
NH2
COO-
COO-
+
NH-Lys252
+
NH
NH
O
approximation
COO-
base catalysis
NH2
(X)3ZnA
base catalysis
strain energy
electrostatic catalysis
COO-
COO-
COO-
COO-
base catalysis
COOB:
:B
H
HO
(X)3ZnA
NH2
N
H
HO
(X)3ZnA
NH2
N+
H
H
base catalysis
HO
(X)3ZnA
NH-Lys252
H
NH2
COO-
N+
H
H
B
acid catalysis