Structure of crystalline α-chymotrypsin: II. A preliminary report including a hypothesis for the activation mechanism

Abstract

An electron density map of tosyl-α-chymotrypsin at 2 A resolution is presented, which shows the conformation of the polypeptide chain. An electron density map of the differences between the tosylated and the native enzyme has also been calculated. These maps lead to the following conclusions. Histidine 57 and serine 195 are known to be part of the active site. In the native enzyme, these are in an environment open to the solvent and in a conformation consistent with the existence of a hydrogen bond between them. In the tosylated enzyme, small movements of histidine 57, serine 195 and methionine 192 bring histidine 57 into a position where it can interact with the sulphonyl group. No other conformational changes are observed. The enzyme contains an ion pair between the α-amino group of isoleucine 16 and the β-carboxyl group of aspartate 194, located in an otherwise non-polar cavity. In conjunction with kinetic and spectroscopic studies (especially Oppenheimer, Labouesse & Hess, 1966) and X-ray analysis of chymotrypsinogen and δ-chymotrypsin at low resolution (Kraut, Sieker, High & Freer, 1962; Kraut, Wright, Kellerman & Freer, 1967), our results lead to a hypothesis for the stereochemistry of the activation process. It is proposed that (i) activation of the zymogen involves no gross reorganization of the main chain nor a significant helix-coil transition; (ii) activation involves a structural change of the enzyme, caused by the formation to an ion-pair between isoleucine 16 and aspartate 194. This structural change would be reversed when the positively charged α-amino group of isoleucine 16 is deprotonated at high pH. The reversal seems to be sterically blocked when the enzyme is inhibited by a bulky group on serine 195. In the light of our model, the location of the disulphide bridges in the chemical sequence of trypsin suggests that trypsin and chymotrypsin have nearly identical tertiary structures, and that their disulphide bridges serve to stabilize rather than to determine the structure. The interactions between the molecules related by the non-crystallographic dyad axes in the crystal are described. One of these involves the active site, but does not inhibit enzymic activity. Of the heavy atoms used for phase determination by the method of isomorphous replacement, platinum(II)- and mercury-containing substituents are bound to sites containing cystine and methionine residues.