Mechanism of Enzymatic Reactions
1. Proteases: involved in many disease states
Heart diseases – thrombin, plasmin, etc.
Alzheimer’s diseases – β and γ-secretases
Osteoporosis – cathepsin K
So on.
a. serine proteases, no cofactors
b. cysteine proteases, no cofactors
c. carboxypeptidases, etc, using Zn2+ as a cofactor
conserved active site
Catalytic triad of serine proteases
Significance: Proteases in blood clotting cascade
Blood clot
Mechanism of chymotrypsinCatalyzed reactions
Catalytic triad
1st phase
2nd phase
TS stabilization
Mechanism of Enzymatic Reactions
2. PLP-dependent enzymes
H
O
O
O
P
O
OH
O
N
CH3
Pyridoxal 5’-Phosphate (PLP)
Enzyme
Enzyme
Assigned reading:
AC Eliot, JF Kirsch, Avoiding the road less
traveled: How the enzyme-substrate
complexes can dictate production selection,
Acc. Chem. Res. 2003, 36, 757-765.
Schiff base formation with external amino acid
2.1. Racemase
L
D-Ala is a component in
peptidoglycan, an indispensable
part of bacterial cell wall.
It is obtained from L-Ala by
racemase.
Mammalian cells don’t have
racemases. Thus, the enzyme
is naturally a target for
antibiotics development.
1.
2.
Walsh CT. 1989. J. Biol. Chem. 264, 2393.
Soda et al. 1986. In “Vitamin B6 pyridoxal phosphate”
Part B, p. 223. Wiley, New York
D
2.2. Decarboxylases
Decarboxylation is irreversible,
Unlike the racemase-catalyzed
reactions
Sukhareva BS 1986. In
“Vitamin B6 pyridoxal phosphate”
Part B, p. 325. Wiley, New York
2.3. Aminotransferases
(Transaminases)
These enzymes are involved
in the degradation of amino
acids, particularly,
the amino moieties.
Transaminases
They are integral part of
the nitrogen cycle in
living systems.
Glutamate dehydrogenase
Glutamate dehydrogenase
Transaminases
Glutamate
dehydrogenase
Urea cycle
Transaminases
In AA degradation
Urea cycle
First half reaction:
H+
×
Deaminate the first substrate
(amino acid) and
Release the first product
(α-keto acid)
Second half reaction:
Aminate the second substrate
(α-keto acid) and
Release the second product
(amino acid)
Equivalent to running the
1st half reaction in reversed
order
2.4. PLP-dependent β-elimination
Involve in degradation of amino acids,
Directly deaminate serine and
threonine.
Irreversible
2.4. PLP-dependent carbon unit transfer
Serine hydroxymethylase
d
X
Carbon unit transfer to tetrahydrofolate (THF) by serine hydroxymethylase
Second half reaction
3. Cytochrome P-450 oxygenases:
Occur in almost every mammalian tissue and organs, as well as in plants,
bacteria, yeast, insects, and so on.
Liver microsomal P450 exist in multiple forms and play important roles in
hydroxylation of endogenous, physiological substrates, as well as a tremendous
ranges of drugs and other chemicals foreign to organisms (called “xenobiotics”)
These enzymes play beneficial roles in detoxifying xenobiotics from the
environment. However, a medicinal chemist has to design the drug candidates
“unattractive” to these enzymes.
Reactions catalyzed
by P450 enzymes
Assigned reading:
Sono M. et al. 1996.
“Heme-containing
Oxygenases” Chem.
Rev., 96, 2841-2887
Reaction mechanisms
Hydroxylation
Epoxidation
Other substrates
containing N, S, etc.
4. Probing enzymatic reaction mechanism
A.
B.
C.
D.
E.
Substrate analogues (molecular probes)
Isotope labeling
Site-directed mutagenesis
Affinity labeling to detect active site residues
NMR/X-ray determination of enzyme, especially
enzyme-substrate(inhibitor) complex.
………………..
Assigned reading:
Newcomb M, Toy PH. 2000. “Hypersensitive radical probes and the mechanisms of cytochrome
P450-catalyzed hydroxylation reactions” Acc. Chem. Res., 33: 449-455
5. Relevance of enzymes to drug development ?
Enzyme
Therapeutic Goal
S-Adenosylhomocysteine hydrolase
Antiviral agent
S-Adenosylmethionine decarboxylase
Antitumor agent
Alanine racemase
Antibacteria agent
D-Amino-acid aminotransferase
Antibacterial agent
G-Aminobutyric acid aminotransferase
Anticonvulsant agent
Arginine decarboxylase
Antibacterial agent
Aromatase
Anticancer agent
Aromatic-L-amino-acid decarboxylase
Synergistic with antiparkinsonian drug
Cysteine proteases
Multiple sclerosis, muscular dystrophy, antibacterial, antiviral agent
Dihydrofolate reductase
Anticancer agent, antibacterial agent, antiprotozoal agent
Dihydroorotate dehydrogenase
Antiparasitic agent, anticancer agent
DNA polymerase I
Antiviral agent
Dopamine β-hydroxylase
Antihypertensive agent, pheochromocytoma agent
α-Glucosidase
Anti-AIDS agent
β-Glucosidase
Diabetes/antiobesity agent
Histidine decarboxylase
Antihistamine, antiullcer agent
β-Lactamase
Synergistic with antibiotics
Lipoxygenase
Anti-inflammatory agent
Monoamine oxidase
Antidepressant agent, antihypertensive agent, antiparkinsonian agent
Ornithine decarboxylase
Anticancer agent, antiprotozoal agent
Ribonucleotide reductase
Antiviral, antitumor agent
Serine hydroxymethylase
Antitumor agent
Serine proteases
Treatment of emphysema, inflammation, arthritis, adult respiratory distress
Testosterone 5α-reductase
Anticancer agent