DIFFERENTIAL CHANGES IN THE ASSOCIATION AND DISSOCIATION RATE CONSTANTS FOR BINDING OF CYSTATINS TO TARGET PROTEINASES OCCURRING ON N-TERMINAL TRUNCATION OF THE INHIBITORS INDICATE THAT THE INTERACTION MECHANISM VARIES WITH DIFFERENT ENZYMES

The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constant s for the binding of recombinant human cystatin C to papain, ficin, ac tinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M(-1).s(-1), whereas the corresponding di ssociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2 ) and 3.5 x 10(-4) s(-1). N-Terminal truncation of the first ten resid ues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant ap prox. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decre ased the association rate constant with cathepsin B approx. 60-fold, w hile minimally affecting the dissociation rate constant. With actinidi n, the truncated cystatin C had both an approx. 15-fold lower associat ion rate constant and an approx. 15-fold higher dissociation rate cons tant than the intact inhibitor. Similar results were obtained for inta ct and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases afte r removal of the N-terminal region is thus due to either a decreased a ssociation rate constant or an increased dissociation rate constant, o r both, depending on the enzyme. This behaviour indicates that the con tribution of the N-terminal segment of the two inhibitors to the inter action mechanism varies with the target proteinase as a result of stru ctural differences in the active-site region of the enzyme.