Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain

J. Simon C. Arthur, John S. Elce

    Research output: Contribution to journalArticlepeer-review

    9 Citations (Scopus)


    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.

    Original languageEnglish
    Pages (from-to)535-541
    Number of pages7
    JournalBiochemical Journal
    Issue number(Pt2)
    Publication statusPublished - 15 Oct 1996


    • Rats
    • Animals
    • Proline
    • Calcium
    • Hydrogen-Ion Concentration
    • Point Mutation
    • Calpain
    • Aspartic Acid
    • Substrate Specificity
    • Protein Conformation


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