3-DIMENSIONAL STRUCTURE OF CHYMOTRYPSIN INACTIVATED WITH (2S)-N-ACETYL-L-ALANYL-L-PHENYLALANYL ALPHA-CHLOROETHANE - IMPLICATIONS FOR THE MECHANISM OF INACTIVATION OF SERINE PROTEASES BY CHLOROKETONES

The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenyla lanyl alpha-chloroethane with gamma-chymotrypsin was studied as a prob e of the mechanism of inactivation of serine proteases by peptidyl chl oroalkanes. It was determined crystallographically that the peptidyl c hloroethane alkylates His57 with retention of configuration at the chi ral center, indicating a double displacement mechanism. We think it li kely that a Ser195-epoxy ether adduct is an intermediate on the inacti vation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 25 6, 481-486] indicate that the epoxy ether intermediate is not an irrev ersibly inactivated form of enzyme [a conclusion confirmed experimenta lly (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both r ing closure of the tetrahedral intermediate to form the epoxy ether an d ring opening by His57 partially limit the first-order rate constant for inactivation, k(i). The peptidyl chloroethyl derivative adopts a v ery different active site conformation from that assumed by serine pro teases inactivated by peptidyl chloromethanes. Positioning the chloroe thyl derivative into the conformation adopted by chloromethyl derivati ves would cause the extra methyl group to make a bad van der Waals con tact with the inactivator P-2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P-1 a mide nitrogen and the carbonyl group of Ser214. We conclude that the u nusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.