THE KINETIC MECHANISM OF SERPIN PROTEINASE COMPLEX-FORMATION - AN INTERMEDIATE BETWEEN THE MICHAELIS COMPLEX AND THE INHIBITED COMPLEX

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1: 1 complexes (denoted EI*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of EI* proceed s via a Michaelis complex (E . I) and so involves at least two steps. In this paper, we determine the rate of EI* formation from alpha-chym otrypsin and alpha(1)-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent deri vative of alpha(1)-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, se rpins are both substrates and inhibitors of the same enzyme. Our resul ts indicate the presence of an intermediate between E . I and EI* and suggest that the partitioning step between inhibitor and substrate pa thways precedes P1-P1' cleavage.