The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the 'occluding loop' of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.

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