title: The kinetic mechanism of serpin-proteinase complex formation. An intermediate between the michaelis complex and the inhibited complex.
abstract: Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) 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 E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-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, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.
authors.affiliation: Department of Chemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: O'Malley
authors.forename: S A
authors.initials: SA
authors.lastname: Nair
authors.forename: H
authors.initials: H
authors.lastname: Rubin
authors.forename: B S
authors.initials: BS
authors.lastname: Cooperman
mesh_terms: D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D015843:Serpins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D015843:Serpins; D010450:Endopeptidases