Chymotrypsin Lecture
Aims: to understand (1) the catalytic
strategies used by enzymes and (2)
the mechanism of chymotrypsin
What’s so great about enzymes?
• They accomplish large rate accelerations
(1010-1023 fold) in an aqueous environment
using amino acid side chains and cofactors
with limited intrinsic reactivity, relative to
catalysts in organic synthesis.
• They are exquisitely specific
Chymotrypsin
• Digestive enzyme secreted by the pancreas
• Serine protease, specific for the peptide
carbonyl supplied by an aromatic residue
(eg Tyr) of a large hydrophobic (eg Met)
Common catalytic strategies
1.
2.
3.
4.
Covalent catalysis
General acid-base catalysis
Metal-ion catalysis
Catalysis by approximation
And enzymes often combine these
strategies eg an example of use of 1 & 2 is
chymotrypsin
Proteases Catalyse a
Fundamentally Difficult Reaction
They cleave proteins by hydrolysis – the
addition of water to a peptide bond
Half life for hydrolysis of typical peptide is 300600 years. Chymotrypsin accelerates the rate of
cleavage to 100 s-1 (>1012 enhancement).
The carbon-nitrogen bond is strengthened by its
double-bond character, and the carbonyl carbon
atom is less electrophilic and is less susceptible to
nucleophilic attack than are the carbonyl carbon
atoms in carboxylate esters.
Identification of the
reactive serine
• Around 1949 the nerve gas di-isopropyl-fluorophosphate
was shown to inactivate chymotrypsin
•
32P-labelled
DIPF covalently attached to the enzyme
• When labelled enzyme was acid hydrolysed the
phosphorus stuck tightly; the radioactive fragment was Ophosphoserine
•
Sequencing established the serine to be Ser195
• Among 28 serines, Ser195 is highly reactive, why?
Probing enzyme mechanism
S1-subsite
Subtilisin
Fig. 1. The isomerization reaction catalyzed by triosephosphate isomerase
Jogl, Gerwald et al. (2003) Proc. Natl. Acad. Sci. USA 100, 50-55
Copyright ©2003 by the National Academy of Sciences
Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 9:
Catalytic Strategies
Copyright © 2007 by W. H. Freeman and Company