The Kinetic Mechanism of Serpin-Proteinase Complex Formation

• Published in: The Journal of biological chemistry 1997-02, Vol.272 (8), p.5354-5359
• Main Authors: O'Malley, Kevin M., Nair, Shrikumar A., Rubin, Harvey, Cooperman, Barry S.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.8.5354

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 α-chymotrypsin and α 1 -antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of α-chymotrypsin with a fluorescent derivative of α 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, 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.