The Kinetic Mechanism of Serpin-Proteinase Complex Formation

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 (E⅐I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from ␣-chymotrypsin and ␣ 1 -antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of ␣-chymotrypsin with a fluorescent derivative of ␣ 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 E⅐I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1 cleavage.1 is a human serine proteinase inhibitor (serpin), a superfamily of proteins believed to have evolved from a common ancestral gene over ϳ500 million years (1-3). The involvement of ACT in Alzheimer's disease (4, 5) and in the regulation of the inflammatory response (6) 2 as well as of prostate-specific antigen activity (8) makes it a particularly interesting protein for study. As is typical of serpins, ACT (I) forms an enzymatically inactive, 1:1 complex (denoted E*I*) with its target proteinases (for example, chymotrypsin) that releases free enzyme and cleaved ACT (I*) only very slowly (9, 10). The complex is designated E*I* to indicate that conformational change has taken place in both the enzyme (10, 11) and inhibitor (12, 13) moieties. For some serpin-proteinase interactions, full inhibition of proteinase requires Ͼ1 eq of serpin. The ratio of moles of inhibitor required per mole of proteinase for 100% inhibition is defined as the stoichiometry of inhibition (SI). Values of SI Ͼ 1 reflect a partitioning of serpin between inhibitor and substrate pathways, giving rise to "suicide inhibitor"-type kinetics (9, 14 -16).A characteristic feature of the serpin-proteinase complex is that it is stable to both heat and SDS treatment, implying covalent bond formation between enzyme and serpin. The nonlability of E*I* may be due either to distortion of the enzyme active site within the complex (10, 11) or to inaccessibility of the covalent E-I linkage toward attacking nucleophilic water, or both. The position of cleavage in released I* occurs within a so-called "reactive center loop," which in intact I extends out from the rest of the molecule, contains a segment of modified ␣-helix (17-21), and is the primary interaction site between the inhibitor and the target proteinase. Following standard nomenclature (22), the position of cleavage takes place between the P1 and P1Ј sites of the inhibitor, which in ACT corr...

Formation of the Covalent Chymotrypsin·Antichymotrypsin Complex Involves No Large-scale Movement of the Enzyme

“…In our own work, both current and previous (5,22,23), on the ACT/Chtr interaction, we have shown that large fluorescent changes, from probes placed at the P7, P13, and P1Ј positions in ACT precede E*I* formation. Similar conclusions from time-resolved studies have been reported for the interactions of antithrombin (AT) and thrombin (14) and antitrypsin and elastase (45).…”

“…Construction, Expression, and Purification of rACTs-S359C-rACT was constructed using sequence overlap expression polymerase chain reaction and the ACT expression vector described previously (20,22). The internal primers coding for the Ser 3 Cys mutation are as follows: 5Ј-CCCTCCTTTGTGCATTAGTGGAGACA-3Ј and 5Ј-GCACAAAGGA-GGGTGATTTTGAC-3Ј (the mutation sites are in boldface).…”

“…Full gene sequencing confirmed a single codon change. S359C-rACT and rACT were purified to homogeneity as described earlier (22).…”

“…Purified protein had a calculated stoichiometry of 1.05 MCM/protein. As shown earlier (22,23), the unique Cys residue in wild-type rACT, Cys 237 , is buried in the hydrophobic core of the molecule and has negligible reactivity under the reaction conditions used for derivatization.…”

“…Rapid Mixing Quenched Flow-Rapid quenched-flow kinetic studies were carried out using a KinTek Chemical-Quench-Flow Model RQF-3 machine followed by densitometric analysis on SDS-PAGE as described previously (22). SDS-PAGE analysis was performed on aliquots of the acid-quenched reaction mixture using gels containing 12% polyacrylamide.…”