In an ongoing study of the mechanisms of calpain catalysis and Ca2+-induced activation, the effects of Asp-104 → Ser and Pro-287 → Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca2+-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10–15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287 → Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104 → Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca2+-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme.

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