Crystal structure of δ-chymotrypsin bound to a peptidyl chloromethyl ketone inhibitor

Chymotrypsin is a member of the trypsin family of serine proteases and is one of the first proteins successfully studied by X-ray crystallography. It is secreted into the intestine as the inactive precursor chymotrypsinogen; four sequential cleavages of the peptide bonds following residues 13, 15, 146 and 148 occur to generate the active π, δ, κ and α forms of chymotrypsin. ¹³C NMR has shown [O'Connell & Malthouse (1995). Biochem. J. 307, 353–359] that when the δ form of chymotrypsin is inhibited by 2-¹³C-enriched benzyloxycarbonylglycylglycylphenylalanyl chloromethane, a tetrahedral adduct is formed which is thought to be analogous to the tetrahedral intermediate formed during catalysis. This inhibitor complex has been crystallized as a dimer in space group P4₁2₁2. The structure has been refined at 2.14 Å resolution to an R value of 21.2% (free R = 25.2%). Conformational differences between δ-chymotrypsin and chymotrypsinogen in the region of the flexible autolysis loop (residues 145–150) were observed. This is the first crystal structure of δ-chymotrypsin and includes two residues which are disordered in previous crystal structures of active chymotrypsin. A difference of 11.3 Ų between the average B values of the monomers within the asymmetric unit is caused by lattice-disordering effects approximating to rotation of the molecules about a crystallographic screw axis. The substrate-binding mode of the inhibitor was similar to other chymotrypsin peptidyl inhibitor complexes, but this is the first published chymotrypsin structure in which the tetrahedral chloromethyl ketone transition-state analogue is observed. This structure is compared with that of a similar tetrahedral transition-state analogue which does not alkylate the active-site histidine residue.