Kinetics of binding of chicken cystatin to papain

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Abstract
The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 .mu.M. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 .times. 106 M-1 s-1 at 25.degree.C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25.degree.C of 31.5 kJ mol-1 and an entrophy of activation at 25.degree.C of -7 jK-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with a excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex. The displaced inhibitor was shown to be intact by several criteria. Analyses of the rate of displacement gave a first-order dissociation rate constant for the complex of .apprx. 5.7 .times. 10-7 s-1 (T1/2 .apprx. 14 days) at 25.degree.C, pH 7.4, I = 0.15, independent of the concentration of the displacing cystatin 1. These findings show that the inhibition of papain by chickens is best described as a simple, reversible bimolecular reaction, leading to formation of an inhibitor-proteinase complex with a dissociation equilibrium constant of .apprx. 60 fM.
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