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/
Nome ncla ture
Enzyme Name s
EC Numb er
Com mon / Recom men ded Name
S yste ma ti c Na me
S yno nyms
CAS Regi str y Num be r
Reacti on & Spec ificity
Pat hway
Cat alys ed Reac ti on
Re ac t ion Type
Nat ural Sub st rates and P ro duct s
S ub st ra t es and P ro duct s
S ub st ra t es
Nat ural Sub st rate
Pro duc ts
Nat ural Pro duc t
Inhibi to rs
Cofacto rs
Met als / Ions
Act iva t ing Comp ou nd s
Ligand s
Funct ional Paramete rs
Km Val ue
Ki Value
pI Val ue
T urnover Numb er
S pecif ic Act ivit y
pH Opt imum
pH Range
Te mp er at ure Opt imum
Te mp er at ure R ange
Is olati on & Preparation
Purificat ion
Clo ned
Re na t ure d
Crys t all iza t ion
Org anis m- rel ated informat ion
Orga nism
S our ce Tiss ue
Loca liza t ion
Stabi lit y
pH St abi lity
Te mp er at ure St abi lity
Ge ner al Stab ilit y
Orga nic Solvent St abil it y
Oxida t ion S t abil it y
S t or age St ab ilit y
Enz yme Struc ture
Se qu ence/ SwissProt li nk
3 D-Str uc tur e/ PDB li nk
Molecular Weight
S ub unit s
Pos ttr an sla ti onal Modif ica t ion
Dise as e & Referenc es
Dis ease
Re feren ces
Applicat ion & Eng ineering
Engineer ing
Appli cat ion
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 - Khne 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: enzyme-
catalyzed 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)
k
1
, also referred to as k
on
, is rate constant for
formation of the E-S complex
k
-1
, also referred to as k
off
, 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 K
s
,
dissociation constant for the E-S complex
K
s
=
E + S E
.
E
.
E + P
k
2
k
-1
k
1
k
-1
k
1
S P
Scheme 1.1
k
on

k
off

Michaelis
complex
When k
2
<< k
-1
,
k
2
called k
cat
(turnover number)
K
s
called K
m
(Michaelis-Menten constant)
Generalized enzyme-catalyzed reaction
k
cat
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 Numbers
a
Enzyme Turnover number
k
cat
(s
-1
)
papain 10
carboxypeptidase
10
2
acetylcholinesterase
10
3
kinases
10
3
dehydrogenases
10
3
aminotransferases
10
3
carbonic anhydrase
10
6
superoxide dismutase
10
6
catalase
10
7
a
Eigen, M.; Hammes, G.G. Adv. Enzymol. 1963, 25, 1.
K
m
is the concentration of substrate that
produces half the maximum rate
K
m
is a dissociation constant, so the smaller
the K
m
the stronger the interaction between E
and S
k
cat
/K
m
is the specificity constant - used to
rank an enzyme according to how good it is
with different substrates
Upper limit for is rate of diffusion (10
9
M
-1
s
-1
)
K
m

k
cat
How does an enzyme release product so
efficiently given that the enzyme binds the
transition state structure about 10
12
times more
tightly than it binds the substrate or products?

After bond breaking (or making) at transition
state, interactions that match in the transition-
state 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)
C
O
O
+
RNH
3
ion-dipole
R
C
NH
3
R'
O
o
o
+
dipole-dipole R
C O
R'
O
o
o
o
o
H
H-bonding
O
RC O H
O
H
charge
transfer
A
D
D
A
hydrophobic
O RC
O
G = -RTlnK
eq

If K
eq
= 0.01, G of -5.5 kcal/mol needed
to shift K
eq
to 100

Specific Forces Involved in
ES Complex Formation
Figure 1.2
NH
3
O
OH
CH
3
COCH
2
CH
2
NMe
3
+
+
o

o
+
o

dipole-dipole
o
+
ion-dipole
O
O
ionic
Examples of ionic, ion-dipole, and dipole-dipole
interactions. The wavy line represents the
enzyme active site
H-bonds
A type of dipole-dipole
interaction between X-H
and Y: (N, O)
Figure 1.3
H-bonds
Hydrogen bonding in the secondary structure of
proteins: o-helix and |-sheet.
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 (AG = AH-TAS)
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 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)
Binding specificity of enantiomers
Scheme 1.2
Enz
L
+ (R,S)
Enz
L
+ Enz
L
R S
diastereomers
Resolution of a racemic mixture
Binding energy for ES complex formation
with one enantiomer may be much higher
than that with the other enantiomer
Both ES complexes may form, but only one
ES complex may lead to product formation
Enantiomer that does not turn over is said to
undergo nonproductive binding
Steric hindrance to binding of enantiomers
Figure 1.4
OOC
NH
3
H
OOC
NH
3
H
A B
S R
Leu
Basis for enantioselectivity in enzymes
Reaction Specificity
Unlike reactions in solution, enzymes can show
specificity for chemically identical protons
Figure 1.5
R
R'
R R'
H
a
H
b
B
-
enzyme
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.
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

Figure 1.6 10
10
-10
14
fold typically
Cat alyzed
Uncat alyzed
React ion Coordinat e
F
r
e
e

E
n
e
r
g
y

(

G
)
A
Uncat alyzed
Enzyme Cat alyzed
React ion Coordinat e
F
r
e
e

E
n
e
r
g
y

(

G
)
B
E+S
E+P
ES
EP
Effect of (A) a chemical catalyst and
(B) an enzyme on activation energy
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
k
non

(s
-1
)
Enzymatic rate
k
cat

(s
-1
)
Rate acceleration
k
cat
/k
non
cyclophilin
a
2.8 x 10
-2
1.3 x 10
4
4.6 x 10
5
carbonic anhydrase
a
1.3 x 10
-1
10
6
7.7 x 10
6
chorismate mutase
a
2.6 x 10
-5
50
1.9 x 10
6
chymotrypsin
b
4 x 10
-9
4 x 10
-2
10
7
triosephosphate
isomerase
b
6 x 10
-7
2 x 10
3
3 x 10
9
fumarase
b
2 x 10
-8
2 x 10
3
10
11
ketosteroid isomerase
a
1.7 x 10
-7
6.6 x 10
4
3.9 x 10
11
carboxypeptidase A
a
3 x 10
-9
578
1.9 x 10
11
adenosine deaminase
a
1.8 x 10
10
370
2.1 x 10
12
urease
b
3 x 10
-10
3 x 10
4
10
14
alkaline phosphatase
b
10
-15
10
2
10
17
orotidine 5'-phosphate
decarboxylase
a
2.8 x 10
-16
39
1.4 x 10
17
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.
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
Scheme 1.3
CH
3
COAr
O O
C
O
C
O
H
3
C
+ CH
3
COO
-
CH
3
+ ArO
-
Second-order reaction of acetate with
aryl acetate
OAr
O
O
O
-
OAr
O
O
O
-
OAr
O
O
O
-
OAr
O
O
O
O
O
-
Relative rate(k
rel
)
1 M
-1
s
-1
220 s
-1
5.1 x 10
4
s
-1
2.3 x 10
6
s
-1
1.2 x 10
7
s
-1
Decreasing rotational and
translational entropy
+ CH
3
COO
-
OAr
Effective Molarity (EM)
5.1 x 10
4
M
2.3 x 10
6
M
1.2 x 10
7
M
220 M
Table 1.3. Effect of Approximation on Reaction Rates
Covalent Catalysis
Scheme 1.4
anchimeric assistance
Most common
Cys (SH)
Ser (OH)
His (imidazole)
Lys (NH
2
)
Asp/Glu (COO
-
)
R Y
O
X
X
R Y
O
-
O R
X
X Z R
O
Activated carbonyl
Z
-
+
1.1
-Y
-
Nucleophilic catalysis
X
-
Scheme 1.5
S
Cl
S
OH
S
+
1.2
HO
-
-Cl
-
Anchimeric assistance by a neighboring group
Model Reaction for Covalent Catalysis
Scheme 1.6
Early evidence to support covalent catalysis
O
O
18
O
O
18
O
CH
3
C
18
O
18
OH
O
OH
18
O
18
+
Ar
H
2
O
H
2
O
O
-
(-ArO
-
)
General Acid/Base Catalysis
This is important for any reaction in which proton
transfer occurs
Figure 1.7 catalytic triad
The catalytic triad of o-chymotrypsin. The
distances are as follows: d
1
= 2.82 ; d
2
=
2.61 ; d
3
= 2.76 .
Scheme 1.7
H
N
N
H
NHR'
Ser
O
H
R
1
O R
2
O
R
N
N H
His
-
OOC Asp
Charge relay system for activation of an active-
site serine residue in o-chymotrypsin
pK
a
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, pK
a
will
rise
Glutamate-35 in the lysozyme-glycolchitin complex
has a pK
a
of 8.2; pK
a
in solution is 4.5
If the carboxylate ion forms salt bridge, it is
stabilized and has a lower pK
a

Basic group in a nonpolar environment has a
lower pK
a

pK
a
of a base will fall if adjacent to other
bases
Active-site lysine in acetoacetate
decarboxylase has a pK
a
of 5.9 (pK
a
in
solution is 10.5)


Two kinds of acid/base catalysis:

Specific acid or specific base catalysis -
catalysis by a hydronium (H
3
O
+
) 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

Figure 1.8
Specific acid/base catalysis General acid/base catalysis
k
k
[Buffer]
[Buffer]
pH 7.9
pH 7.3
pH 7.9
pH 7.3
A
B
Effect of the buffer concentration on (A)
specific acid/base catalysis and (B)
general acid/base catalysis
Scheme 1.8
Specific Acid-Base Catalysis
O
C OEt
poor
nucleophile
weak
electrophile
+ +
H
3
C EtOH CH
3
COOH H
2
O
Hydrolysis of ethyl acetate
Scheme 1.9
Alkaline hydrolysis of ethyl acetate
O
C
OH H
3
C
O
C
OC
2
H
5
H
3
C
O
C
O
-
H
3
C
+
+
strong
nucleophile
C
2
H
5
O
-
HO
-
C
2
H
5
OH
Scheme 1.10
O
C
OH H
3
C
OH
C
OC
2
H
5
H
3
C
OH
C
OC
2
H
5
H
3
C
O
C
OC
2
H
5
H
3
C
+
+
+
+
strong
electrophile
H
3
O
+
H
2
O
C
2
H
5
OH
Acid hydrolysis of ethyl acetate
Scheme 1.11
B
+
H
R Y
O
H OH
B:
Simultaneous acid and base enzyme catalysis
base catalysis
acid catalysis
Enzymes can utilize acid and base
catalysis simultaneously
Simultaneous acid/base catalysis is the reason for
how enzymes are capable of deprotonating weak
carbon acids
Scheme 1.12
Simultaneous acid and base enzyme catalysis
in the enolization of mandelic acid
Ph
H
a
HO
OH
b
O
Ph
O
-
O
H
a
HO
Ph
H
a
HO
OH
b
OH
c
Ph
H
a
HO
OH
b
OH
c
Ph
HO
O
-
OH
b
Ph
HO
OH
c
OH
b
pK
E
= 18.6
+
+
pK
a
~ 7.4
pK
a
= 6.6
H
c
+
H
a
+
pK
E
= 15.4
pK
a
= 22.0
H
a
+
pK
a
~ -8
H
c
+
pK
a
= 3.4
H
b
+
1.3
1.4
1.5
1.6
Low-barrier hydrogen bonds - short (< 2.5),
very strong hydrogen bonds
Stabilization of the enolic intermediate occurs
via low-barrier hydrogen bonds
X R
H
O
H
B:
B H
X R
H
O
:B
B
+
H
R
O
R = H, alkyl, SR'
O

O X
H H
M
+
B H
O

O X
H
M
+
B
O
O
H
M
+
B
H
B
H
B
B:
B:
BH
-HX
A
B
Scheme 1.13
low-barrier
H-bond
weak
base
strong
acid
strong
base
weak acid
low-barrier
H-bond
stronger acid
needed
One-base
mechanism
syn-elimination
carboxylic acids
Simultaneous acid and base enzyme catalysis in the
1,4-elimination of |-substituted (A) aldehydes,
ketones, thioesters and (B) carboxylic acids
Two-base
mechanism
anti-elimination
Scheme 1.14
ElcB mechanism - not relevant
X R
H
O
H
X R
O
B
+
H
R
O
B:
Base catalyzed 1,4-elimination of |-substituted
carbonyl compounds via an enolate
intermediate (ElcB mechanism)
Needs acid or metal catalysis
Alternative to Low-Barrier Hydrogen Bond
Scheme 1.15
R
H
O
H
R' R
O
R'
H
B:
B
+
H
Electrostatic enzyme catalysis in enolization
Electrostatic Catalysis
Scheme 1.16
oxyanion hole
H
N
N
H
H
N
N
H
N
H
H
N
N
H
H
N
O
O
O O
O
O
O
R"
R'
R R"
R'
R
O
+
+
also could be a
H bond or dipole
Electrostatic stabilization of the transition state
Desolvation
Exposes substrate to lower dielectric
constant environment
Exposes water-bonded charged groups for
electrostatic catalysis
Destabilizes the ground state
The removal of water molecules at the active site
on substrate binding
Scheme 1.17
Strain Energy
k
1.8

k
1.7

= 10
8

O
P
O
HO
-
O
P
O
O
- O
O
-
-
O
O
P
O
O
- -
O
CH
3
CH
3
O
P
O
-
O
-
O
CH
3
CH
3
HO
1.7
1.8
-
OH
-
OH
Alkaline hydrolysis of phosphodiesters
Figure 1.9
Induced Fit Hypothesis
putting strain energy into the substrate
Figure 1.10
Energetic Effect of Enzyme Catalysis
Importance of ground state destabilization
H
Lys
252
NH
2
NH
2
O
COO
-
NH
2
O
COO
-
NH
NH-Lys
252
COO
-
NH
2
COO
-
B:
NH
NH-Lys
252
COO
-
NH
2
COO
-
N
H
COO
-
COO
-
H
NH
2
NH-Lys
252
B
B:
H
:B
N
H
COO
- COO
-
NH
2
H
N
H
COO
- COO
-
NH
2
+
+
..
+
+
+
..
:
Zn
B
(Cys)
4
Lys
252
NH
NH
2
O
COO
-
Zn
B
(Cys)
4
H
:B
Lys
252
NH
NH
2
OH
COO
-
Zn
B
(Cys)
4
Lys
252
NH
H
2
N
COO
-
NH
2
O
COO
- Lys
252
NH
N
COO
-
H
H
:B
(X)
3
Zn
A
HO
(X)
3
Zn
A
HO
H
(X)
3
Zn
A
HO
(X)
3
Zn
A
HO
(X)
3
Zn
A
HO
strain energy
electrostatic catalysis
approximation
covalent catalysis
base catalysis
strain energy
electrostatic catalysis
base catalysis
base catalysis
acid catalysis
base catalysis
base catalysis
approximation
approximation
(X
3
)Zn
A
(X
3
)Zn
A
Mechanisms of Enzyme Catalysis - porphobilinogen synthase