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 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.
O'Malley
K M
KM
Department of Chemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Nair
S A
SA
Rubin
H
H
Cooperman
B S
BS
D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D015843:Serpins / Q000378:metabolism*; D013379:Substrate Specificity
D015843:Serpins; D010450:Endopeptidases