Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Peptide Production 279

A

Asp- ,,o C-O'- .......... H_NT. H-O-Ser 195

102

\ /

i c c I

H His 57

I

B o

II

Asp-- C-- O7--'+ H- N N'H

\ /

/c c

I

H His 57

C

O- Ser 195

Substrate

Fig. 1. The catalytic triad at the active-site of chymotrypsin. (A) Enzyme alone.
(B) On addition of substrate, a proton is transferred from serine 195 to histidine 57,
resulting in the formation of a strongly nucleophilic oxyanion on Ser 195. The
positively charged imidazole ring generated in this process is stabilized by electro-
static interaction with the negatively charged Asp 102. The oxyanion carries out
nucleophilic attack on the peptide bond.

as a serine protease, since its catalytic activity results from a strongly
ionized serine residue that is generated at the active site as a consequence
of the microenvironment of serine residue 195 (see Fig. 1). This strongly
nucleophilic oxyanion catalyzes the cleavage of peptide bonds. Many
inhibitors of serine proteases (such as diisopropylphosphofluoridate [DFP]
and phenylmethane sulfonyl fluoride [PMSF]) function by reacting with
the active site serine residue. The commercially available source of the
enzyme is from bovine pancreas (3-5).

2.1.2. Specificity
Chymotrypsin has relatively broad specificity, cleaving on the C-
terminal side of hydrophobic residues, especially phenylalanine, tyrosine,
tryptophan, and leucine. However, some significant cleavages have