Antichymotrypsin interaction with chymotrypsin: Identification of intermediates on the path to inhibited complex

Serine proteinase inhibitors (serpins) form enzymatically inactive 1:1 complexes with their target proteinases that are generally long lived. This stable complex, designated E*I*, is most likely an acyl enzyme, analogous to the intermediate from substrate turnover. The focus of this thesis is to show evidence for intermediates on the path from encounter complex to E*I* formation and to describe the pathway in terms of a kinetic scheme. The interaction is investigated by the use of site specific fluorescent probes on both serpin and proteinase in combination with the rapid kinetic techniques of stopped flow and quenched flow. This allows for the observation of transient species both by probe sensitivity to environmental changes and the use of fluorescence resonance energy transfer (FRET). Fluorescent labeling of a Cys variant of rACT at position P7 of the reactive center loop (RCL) with 4-bromomethyl-7-methoxycoumarin (BNMC) yields a derivative that inhibits α-chymotrypsin in the normal manner and can be used to follow the serpin: proteinase interaction by stopped flow spectrofluorometry. At 40°C, the saturated rate of the observed fluorescence change upon mixing the derivative with α-chymotrypsin exceeds the saturated rate of E*I* formation as measured by quenched flow. This provides kinetic evidence for an intermediate between encounter complex and inhibited complex and also suggests that the P1-P1′ linkage remains intact in the observed intermediate. Derivatization of both serpin and proteinase with fluorophores allows for the reaction to be monitored by FRET as well as by simple changes in fluorescence. A Cys variant of rACT at P1′ was derivatized with BMMC. Chymotrypsin was also labeled with the succinimidyl ester of 7-diethylaminocoumarin-4-acetic acid specifically at its N-terminus. The fluorescently labeled serpin:proteinase pair retains a Stoichiometry of Inhibition (SI) of 1, and second order inhibition rate constants show only a minor reduction, as compared with the unlabeled pair, resulting from incorporation of labels on each protein. The double labeled system also forms an effective donor:acceptor pair. Changes in the distance between labeled chymotrypsin and P1′ residues are observable by FRET. At least three intermediates are directly observed, with the interprobe distance decreasing as the reaction moves toward stable complex formation. The direct conclusion from this result is that no major translocation of the enzyme takes place upon stable complex formation