The Organic Chemistry of
Drug Design and Drug
Action
Chapter 4
Enzymes
Enzymes
Just like drug-receptor complexes, enzymes
form complexes with substrates.
Two characteristics of enzymes:
• recognize a substrate
• catalyze a reaction with it
Enzymes - proteins that catalyze reactions in a
biological system. They function by lowering transition
state energies and by raising ground state energies.
Fischer(1894) – lock and key
Henri and Brown (1902) – ES complex is formed
Pauling (1946) - Enzyme is a flexible template designed to be
complementary to structure of substrate at the transition state of the
reaction, not the ground state.
As the reaction proceeds toward the transition state, the enzyme
interacts more effectively, which accelerates the reaction - called
transition state stabilization.
Conformational change occurs to align catalytic groups and destabilize
the enzyme – Koshland-induced fit hypothesis.
Similar to noncatalytic receptors, substrate binds to
a small part of enzyme - called the active site.
Two key factors of enzyme catalysis:
• specificity
• rate acceleration
Active site contains amino acid side chains used in
catalysis.
Some enzymes require cofactors (also called
coenzymes):
• organic molecules
• metal ions
Specificity of Enzyme
Reactions
specificity of binding
specificity of reaction
Enzyme catalysis initiated by formation of ES complex
(Michaelis complex)
Involve same interactions as drug-receptor complex
Scheme 4.1
Michaelis
complex
Transition state
Binding specificity can be absolute (only one substrate)
or very broad (many substrates).
Enzymes are chiral catalysts (all L-amino acids)
Interaction with a racemic mixture gives two
diastereomeric complexes
Therefore, different energies, different reactivities.
Both ES complexes may form, but only one may lead to
product.
Resolution of Racemic Mixtures
with Chiral Reagents
Scheme 4.2
H3 C
H
Ph
CH3
+
COOH
NH2
4.2
4.1
H CH3
H3 C
H
Ph
NH3+ -OOC
+
H3 C H
H3 C
H
Ph
NH3+ -OOC
(R.S)
(R.R)
Diastereomeric salts
FIGURE 4.1 Differential binding
interactions by enantiomers. (A)
binding pocket for the (S)-isomer; (B)
steric hindrance with the (R)-isomer.
Reaction specificity depends on
• Acidic, basic, and nucleophilic groups of the
active site amino acids
• Coenzymes (also called cofactors)-specific
organic molecules or metal ions
Specificity for chemically
identical protons
Figure 4.2
Only Ha is removed stereospecifically
Rate Acceleration
Catalysts stabilize transition state energy relative to
ground state, which decreases G‡ (Ea)
Chemical catalyst
Enzyme
Figure 4.3
Enzyme stabilizes TS‡, destabilizes ES, destabilizes
intermediates.
Mechanisms of Enzyme
Catalysis
Most common:
• Approximation,
• Covalent catalysis,
• General acid-base catalysis,
• Electrostatic catalysis,
• Desolvation,
• And strain
Approximation
• Rate acceleration by proximity
• After the ES complex forms, the reaction
becomes 1st order rather than 2nd order.
• Equivalent to increasing the concentration of
the reacting groups.
Consider a 2nd order reaction
Scheme 4.3
Model for Approximation
Effective molarity
(EM) - concentration
of the catalytic group
required to cause the
intermolecular
reaction to proceed at
the observed rate of
the intramolecular
reaction.
Table 4.1
Covalent Catalysis
Scheme 4.4
Enzymatic analogy to anchimeric assistance (neighboring
group assistance)
Common active site nucleophiles:
- SH (Cys)
N
N
H
(His)
- OH (Ser)
- NH2 (Lys)
- COOH (Glu/Asp)
Anchimeric assistance by a
neighboring sulfur atom
Scheme 4.5
General Acid-Base Catalysis
Important whenever proton transfer occurs
Two kinds of acid-base catalysis: specific and
general
Specific acid-base catalysis is determined by
[H3O+] or [HO-] (i.e., the pH).
General acid-base catalysis occurs by an increase
in buffer concentration at a constant pH.
Specific Acid-Base Catalysis
Consider
poor electrophile
Scheme 4.6
poor
nucleophile
Increase pH
Scheme 4.7
excellent nucleophile
Decrease pH
Scheme 4.8
excellent electrophile
General Acid-Base Catalysis
Unlike reactions in solution, enzymes can use
acid and base catalysis simultaneously.
Scheme 4.9
general acid
general base
pKa values of amino acids in the active site are
not necessarily the same as in solution
Example of General Acid-Base
Catalysis
Scheme 4.10
-chymotrypsin
Electrostatic Catalysis
oxyanion hole
Scheme 4.11
transition state
stabilization
Desolvation
Desolvation (removal of H2O molecules) of
charged groups destabilizes the ground state
(less polar environment); therefore groups are
more reactive.
Also, after desolvation, the charged groups in
Scheme 4.11 are more able to stabilize charge in
the transition state.
Strain or Distortion
Scheme 4.12
Because of ring strain, 4.4 is much more reactive
than 4.5.
Strain induced in the enzyme or in the substrate
raises the ground state energy (induced fit theory).
Conformational change may be used to induce
strain energy.
Destabilization of the ground state is necessary to assure
smaller activation energies.
Figure 4.4
Rate accelerations of 1010 - 1014 times nonenzymatic
reactions.
Product release sometimes is slow step.
Example of Enzyme Catalysis
peptidoglycan transpeptidase
Scheme 4.13
Coenzymes (or Cofactors)
• Organic molecules or metal ions essential
for catalytic action
• Most organic cofactors are derived from
vitamins (essential nutrients).
• Only coenzymes whose chemistry will be
utilized throughout the remainder of the
course are discussed.
Enzyme cofactors
Thiamine
Riboflavin
Enzyme cofactors
NAD(P)+
NAD(P)H
Enzyme cofactors
Pyridoxal-5’phosphate
Cobalamin
Enzyme Cofactors
Folic acid
Tetrahydrofolate
Enzyme cofactors
Biotin
Pantothenic acid
Coenzyme A
Enzyme cofactors
Heme
Glutathione
Enzyme cofactors
Adenosine triphosphate Lipoic acid
(ATP)
Ascorbic
acid
Coenzymes and vitamins
Diseases caused by vitamin deficiency
Pyridoxal 5’-phosphate (PLP)
(from vitamin B6)
Catalyzes
reactions of
amino acids
O
H
OH
=O PO
3
N
CH3
PLP
PLP bound at
active site
FIGURE 4.5 Pyridoxal 5′-phosphate covalently bound to the active
site of an enzyme.
abbreviated
structue
First Step in All PLP-Dependent Reactions
Scheme 4.14
From here all of the PLP reactions occur
Pyridinium group can stabilize electrons by
resonance from the C-H, C-COO-, or C-R bonds.
To get regiospecific cleavage the bond that
breaks must lie in a plane perpendicular to the
plane of the PLP-imine -e- system.
Figure 4.6
C-H bond is perpendicular to the
 system
How do you freeze free rotation and control which
bond is perpendicular?
Dunathan Hypothesis (1971)
A cationic group could interact with the
carboxylate to control the positions of the bonds
perpendicular to the -system.
FIGURE 4.7 Dunathan hypothesis for PLP activation of the Cα–N bond. The
rectangle represents the plane of the pyridine ring of the PLP. The angle of sight is
that shown by the eye in Figure 4.6. From Dunathan, H. C. (1971) In “Advances in
Enzymology,” Vol. 35, p. 79, Meister, A., Ed. Copyright © 1971. This material is
reproduced with permission of John Wiley & Sons, Inc.
PLP
Racemases
Scheme 4.15
All steps are reversible
Keq = 1
Decarboxylases
Scheme 4.16
Aminotransferases
(formerly called transaminases)
Scheme 4.17
First Half Reaction of
Aminotransferases
Scheme 4.18
Second Half Reaction of Aminotransferases
Scheme 4.19
-Elimination
When X is a
leaving group,
elimination can
occur.
Scheme 4.20
Tetrahydrofolate and
Pyridine Nucleotides
Coenzymes that transfer one-carbon units are
derived from the vitamin folic acid.
Reduction of two C=N double bonds to give
tetrahydrofolate requires another coenzyme,
reduced nicotinamide adenine dinucleotide
(NADH).
tetrahydrofolate
Reduced nicotinamide adenine dinucleotide
coenzymes are derived from vitamin B3 (niacin).
part vital to the chemistry
H O
H
NH2
N
When R’ = H
When R’ = PO3=
NADH
NADPH
R
abbreviated structure
NAD(P)H reacts like a hydride reducing agent to reduce the imine
bonds.
Scheme 4.21
folic acid
dihydrofolate
tetrahydrofolate
Tetrahydrofolate is involved in reactions that transfer one-carbon
units from one substrate to another.
Different pyridine nucleotide enzymes use only one
of the two hydrogens at C-4. Because the enzyme
can differentiate these chemically equivalent
hydrogens, they are named differently.
An atom is prochiral if by changing one of its
substituents, it is converted from achiral to chiral.
Prochiral H’s - if HR is changed to 2H, then the
carbon becomes chiral (R stereochemistry)
pro-S hydrogen
pro-R hydrogen
The pro-R and pro-S protons of NAD(P)H
are diastereotopic
The carbon atom that
is transferred is
derived from serine in
a PLP-dependent cleavage reaction.
Scheme 4.22
atom to be
transferred
Because of the toxicity of formaldehyde, the reaction in Scheme
4.22 does not occur until the acceptor (tetrahydrofolate) is bound.
Scheme 4.23
N10-methylenetetrahydrofolate
N5 -methylenetetrahydrofolate
N5 ,N10-methylenetetrahydrofolate
(Keq = 3.2 x 104)
These forms of the coenzyme can transfer a hydroxymethyl group to substrate.
N5 ,N10-Methylenetetrahydrofolate can be oxidized by a NADP+dependent enzyme to give N5 ,N10-methylenyltetrahydrofolate.
Scheme 4.24
hydrolysis
gives
N10-formyltetrahydrofolate
N5-formyltetrahydrofolate
N5 ,N10-Methylenetetrahydrofolate can be reduced by an
NADPH enzyme to give N5-methyltetrahydrofolate.
Scheme 4.25
Biosynthesis of Purines
Example of a one-carbon transfer at the formate oxidation state.
Scheme 4.26
inosine MP
Flavin Coenzymes
Derived from riboflavin (vitamin B2; 4.49)
Flavin mononucleotide (FMN)
4.50
Flavin adenine dinucleotide (FAD)
4.51
Flavin
Reactions
Table 4.5
Scheme 4.27
oxidized
semiquinone form
reduced
Conversion of reduced flavin back to oxidized
flavin can involve one of two different
mechanisms.
Scheme 4.28
Flavoenzymes that require a one-electron acceptor
(ubiquinone or cytochrome b5) are called
dehydrogenases.
Flavoenzymes that utilize O2 are called oxidases.
Scheme 4.29
Example of a Two-Electron Flavoenzyme Reaction
D-Amino acid oxidase
Scheme 4.30
Example of Carbanion Followed by Two OneElectron Transfers
Scheme 4.31
Example of Two One-Electron Transfer Mechanism
Monoamine oxidase
(MAO)
Scheme 4.32
Example of a Hydride Mechanism
Uridine diphosphate N-acetylenolpyruvylglucosamine
reductase
Scheme 4.33
requires NADPH to
reduce the flavin
Flavin monooxygenases - important in drug
metabolism; incorporate an oxygen atom from O2
into substrate.
Scheme 4.34
flavin
4-hydroperoxide
Heme
(protoporphyrin IX)
Cofactor in cytochrome P450 family of enzymes;
important in drug metabolism - hydroxylations and
epoxidations.
Mechanism for P450-Catalyzed
Hydroxylation
Scheme 4.35
high-energy iron-oxo species
Some General P450
Mechanisms
Scheme 4.36
Hydroxylation
radical lifetime is very short
Scheme 4.37
Epoxidation
Scheme 4.38
Sulfoxidation
Figure 4.9
ATP activates low reactivity molecules
What if you mixed benzoic acid
with ammonia? You would not
get the amide.
Scheme 4.39
To get the amide, first
activate the carboxylic acid.
Scheme 4.40
ATP activates molecules by acting like
acetic anhydride or thionyl chloride.
Conversion
of fatty acids
to fatty acylcoenzyme A
derivatives
Scheme 4.41
Enzymes are useful to make drugs, since they
have high substrate specificity and
stereospecificity
Chemical synthesis of Lyrica
Key step in enzymatic synthesis of Lyrica
Sitagliptin, an antidiabetic drug
Enzymatic synthesis of sitagliptin
Enzyme Therapy
Mostly hydrolytic reactions
lactase, amylase, ligase, cellulase, trypsin, papain,
pepsin - for GI disorders
asparaginase - leukemia
urokinase, streptokinase, and tissue plasminogen
activator (tPA) convert plasminogen to plasmin,
an enzyme that digests blood clots
Drawbacks - protease degradation and allergic
responses