y-Chymotrypsin Is a Complex of a-Chymotrypsin with Its Own

The determination of three separate y-chymotrypsin structures a t different temperatures and resolutions confirmed the presence of electron density in the active site, which could be interpreted as an oligopeptide as had previously been suggested by Dixon and Matthews [( 1989) Biochemistry 28,7033-70381. HPLC analyses of the enzyme before and after crystallization demonstrated the presence of a wide variety of oligopeptides in the redissolved crystal, most with COOH-terminal aromatic residues, as expected of the products of chymotrypsin cleavage, which appeared to arise from extensive autolysis of the enzyme under the crystallization conditions. The refined structures agree well with the conformation of both y-chymotrypsin and a-chymotrypsin. The electron density in the active site is thus interpreted as arising from a repertoire of autolysed oligopeptides produced concomitantly with crystallization. The COOH-terminal carbons of the polypeptide(s) display short contact distances (1.97, 2.47, and 2.13 A, respectively) to Ser195 0 7 in all three refined structures, but the electron density is not continuous between these two atoms in any of them. This suggests that some sequences are covalently bound as enzyme intermediates while others are noncovalently bound as enzyme-product complexes. x e serine protease y-chymotrypsin (7-Cht)' was identified as a crystalline form distinct from a-chymotrypsin (a-Cht) This work was supported by US. Army Medical Research and Development Command Contracts DAMD17-GC-7037 and DAMD17-89C-9063, by the Minerva Foundation, Munich, Germany, and by the US.-Israel Binational Science Foundation. 3 Coordinates (code name 8GCH) for y-chymotrypsin (Nat-I) were deposited in the Protein Data Bank, Brookhaven National Laboratory (Bernstein et al., 1977). * Correspondence should be addressed to these authors. I Department of Neurobiology. Department of Structural Chemistry. Department of Chemical Services. by Kunitz (1938) although it has a very similar conformation (Matthews et al., 1967; Cohen et al., 1981). Although a-Cht and y-Cht are believed to be identical in primary sequence (Desnuelle, 1960), the question of whether the two crystalline forms originate from two distinct conformational species in solution has remained a matter for discussion (Corey et al., 1965; Dixon & Matthews, 1989). y-Cht is crystallized at higher pH (e.g., 5.6 vs 4.2) than a-Cht and packs as a mol Abbreviations: a-Cht, athymotrypsin; Cht, chymotrypsin; Chtph7, y-Cht at pH 7.0; y-Cht, y-chymotrypsin; OMTKY3, turkey ovomucoid inhibitor third domain; TFA, trifluoroacetic acid. 0006-2960/91/0430-5217%02.50/0