The N-terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two-step binding by rapid-kinetic studies of cystatin A labeled at the N-terminus with a fluorescent reporter group

The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops farming the remainde r of the inhibitory surface. In an attempt to demonstrate such two-step bin ding, which could not be identified in previous kinetics studies, we introd uced a cysteine residue before the N-terminus of cystatin A and labeled thi s residue with fluorescent probes. Binding of AANS- and AEDANS-labeled cyst atin A to papain resulted in similar to4-fold and 1.2-fold increases of pro be fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measur ed by the loss of papain activity in the presence of a fluorogenic substrat e, for the reaction of the enzyme with excess AANS-cystatin A increased lin early with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluoresc ence with either excess enzyme or labeled inhibitor, showed an identical hy perbolic dependence on the concentration of the reactant in excess. This de pendence demonstrates that the binding occurs in two steps, and implies tha t the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affini ties and dissociation rate constants for the binding of labeled and unlabel ed cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a sim ilar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteris tic of most cystatin-proteinase reactions.