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/
Nomenclature
Enzyme Names
EC Number
Common/ Recommended Name
Systematic Name
Synonyms
CAS Registry Number

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

Reaction & Specificity

Pathway
Catalysed Reaction
Reaction Ty pe
Natural Substrates and Products
Substrates and Products
Substrates
Natural Substrate
Products
Natural Product
Inhibitors
Cofactors
Metals/ Ions
Activating Compounds
Ligands
Isolation & Preparation
Purifcation
Cloned
Renatured
Crystallization

Stability
pH Stability
Te mperature Stability
General Stability
Organic Solvent Stability
Oxidation Stability
Storage Stability
Disease & References
Disease
References

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: enzymecatalyzed reaction always initiated by the
formation of an enzyme-substrate (or ES)
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 ES 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 (ES complex)
k1, also referred to as kon, is rate constant for
formation of the ES complex
k-1, also referred to as koff, is rate constant for
breakdown of the complex
Stability of ES complex is related to affinity of
the substrate for the enzyme as measured by Ks,
dissociation constant for the ES complex

Generalized enzyme-catalyzed reaction

E+S

kon
k1
k1
koff
k1
Ks=
k1

Michaelis
complex

E .S

k2

E .P

E+P

Scheme 1.1

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
acetylcholinesterase
kinases
dehydrogenases
aminotransferases
carbonic anhydrase
superoxide dismutase
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)

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

Specific Forces Involved in

ES Complex Formation
Examples of ionic, ion-dipole, and dipole-dipole
interactions. The wavy line represents the
enzyme active site

NH

+
CH

COCH

CH

NMe

OH

Figure 1.2

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-TS)

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

Resolution of a racemic mixture

EnzL+(R,S)
Scheme 1.2

EnzL

EnzL

diastereomers

 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

Basis for enantioselectivity in enzymes
A

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

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'

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

Effect of (A) a chemical catalyst and

(B) an enzyme on activation energy
A

Uncatalyzed

(G)

Uncatalyzed

FreeEnergy(G)

Catalyzed

EnzymeCatalyzed

E+S

ES

EP

E+P

ReactionCoordinate

Figure 1.6

ReactionCoordinate

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

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

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

CH3

+ArO

Table 1.3. Effect of Approximation on Reaction Rates

Relativerate

)
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

Anchimeric assistance by a neighboring group

S

Cl

Cl

+
S
1.2

Scheme 1.5

HO

OH

Model Reaction for Covalent Catalysis

Early evidence to support covalent catalysis
O

18O

O Ar

18

CH3C O
H2O
(ArO)

Scheme 1.6

O
18O
18O

H2O

18O
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 ; d2 =
2.61 ; d3 = 2.76 .

catalytic triad

Figure 1.7

Charge relay system for activation of an activesite serine residue in -chymotrypsin

H
N

NHR'

N
H

R1
Ser

R2

H
N

N
His

Scheme 1.7

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

pH7.9

pH7.9

pH7.3

pH7.3

[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

+ H2 O
poor
nucleophile

CH3COOH

EtOH

Alkaline hydrolysis of ethyl acetate

O
H 3C

O
OC2H5

HO
strong
nucleophile

Scheme 1.9

H3C

O
OH

C2H5O

H3C

C2H5OH

Acid hydrolysis of ethyl acetate

+OH

O
H3C

OC2H5

+ H3

O+

H3C

OH
OC2H5

C
H3C + OC2H5

strong
electrophile

Scheme 1.10

H2O
H3C

OH

+ C2H5OH

Enzymes can utilize acid and base

catalysis simultaneously
Simultaneous acid and base enzyme catalysis

O
R

B:

B+

Y
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

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

 Low-barrier hydrogen bonds - short (< 2.5),

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

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

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

B:

Scheme 1.14

O
R

H
B+

Alternative to Low-Barrier Hydrogen Bond

Electrostatic enzyme catalysis in enolization

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

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
covalentcatalysis

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