Fundamentals of Biochemistry

Biochem 380 - Fall 2006

We'll finish chapter 6 with a detailed look at the catalytic mechanism of chymotrypsin, an enzyme belonging to the family of serine proteases
These enzymes catalyze the cleavage of a peptide bond, using a serine residue acting as one of the key catalytic residues in the active site
The serine, in conjunction with a histidine and an aspartate, form a catalytic triad, the three main residues that are involved in the reaction mechanism
The mechanism of chymotrypsin makes use of all of the catalytic modes introduced in chapter 6:
- the chemical effects of acid-base catalysis and covalent catalysis
- the binding effects of proximity and transition state stabilization

Three different serine proteases are shown above, each cleaving a different type of substrate.
All three enzymes have a two-lobed structure with the active site in the cleft between. The residues that form the catalytic triad are shown in red
These enzymes are all synthesized in an inactive form as zymogens, and then activated by proteolytic cleavage of a small portion of the polypeptide chain
This produces a conformational change in the structure, opening up a hydrophobic substrate-binding pocket.

The specificity of each enzyme is determined by the size and character of its substrate-binding site
Chymotrypsin cleaves on the carboxyl side of bulky hydrophobic residues such as phenylalanine and tyrosine, and has a hydrophobic pocket that supports the binding of these residues
Trypsin cleaves at positively-charged lysine and arginine residues, which is promoted by a negatively-charged aspartate residue at the bottom of its substrate-binding site
Elastase degrades elastin, a fibrous protein rich in glycine and alanine residues. Cleavage at these smaller residues occurs in a correspondingly smaller binding pocket in the enzyme

The three catalytic residues in the active site of chymotrypsin are an aspartate (Asp-102), a histidine (His-57) and a serine (Ser-195)
The three residues are positioned in a way that promotes the creation of a powerful nucleophile on the serine side chain
Upon compression, the negative charge of the aspartate increases the pKa of the histidine from 7 to around 11
This increased basicity allows the histidine to abstract a proton from the serine, generating a highly-reactive alkoxide ion