Antichymotrypsin Interaction with Chymotrypsin

Abstract

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9) and of the serpin molecule in general. The "hinge" region of the reactive center loop, including residues P10 -P14, shows facile movement in and out of ␤-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the ␣ 1 -antichymotrypsin⅐␣-chymotrypsin encounter complex, E⅐I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of ␣ 1 -antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates,

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).…”

“…In Luo et al (23), the time dependence of fluorescent change and of E*I* formation, measured at pH 7 and 10°C, could be adequately fit with three rate constants, and it was only in considering all of the rate data collected over the pH range of 5.0 to 8.0 that the need for four rate constants became clear. Thus, it is appropriate to compare rate constants k 1 (27.5 s Ϫ1 ), k 2 (2.5 s Ϫ1 ), and k 3 (0.6 s Ϫ1 ) measured in this work with rate constants k 1 (75 s Ϫ1 ), k 2 (5.6 s Ϫ1 ), and k 3, app (0.7 s Ϫ1 ), respectively, measured in Luo et al, where k 3, app , the overall rate constant for conversion of EI b to E*I*, is equal to k 3 k 4 /(k 3 ϩ k 4 ).…”

“…Kinetic Scheme-Recent pH-dependent studies of the reaction of a fluorescent derivative of a cysteine variant of rACT at the P13 position with Chtr enabled us to formulate a scheme for rACT interaction with Chtr (Scheme 2), similar to Scheme 1 but containing three intermediates between E⅐I and E*I* (23). The nature of the pH dependence of the fluorescent changes and the similarity of Scheme 2 to the interaction of substrates with Chtr (40,41) led us to propose that E⅐I conversion to EI a principally involves rearrangement of the RCL to the required canonical conformation.…”

“…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.…”

“…Inhibition rate constants were determined by incubating equimolar concentrations of enzyme and inhibitor under second-order conditions and removing aliquots for residual enzyme activity determination as described earlier (20). SI values were determined by densitometric analysis of SDS-PAGE gels by comparing the intensity of cleaved serpin following complex formation with proteinase with the band intensity of unreacted intact serpin (23).…”