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 journalArticle

    9 Citations (Scopus)

    Abstract

    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
    Volume319
    Issue number(Pt2)
    Publication statusPublished - 15 Oct 1996

    Fingerprint

    Calpain
    Proline
    Aspartic Acid
    Catalytic Domain
    Autolysis
    Papain
    Mutation
    Enzymes
    Chemical activation
    Ions
    Enzyme Stability
    Enzyme Activation
    Cysteine Proteases
    Catalysis
    Serine
    Conformations
    m-calpain

    Keywords

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

    Cite this

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    abstract = "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.",
    keywords = "Rats, Animals, Proline, Calcium, Hydrogen-Ion Concentration, Point Mutation, Calpain, Aspartic Acid, Substrate Specificity, Protein Conformation",
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    Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain. / Arthur, J. Simon C.; Elce, John S.

    In: Biochemical Journal, Vol. 319, No. (Pt2), 15.10.1996, p. 535-541.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain

    AU - Arthur, J. Simon C.

    AU - Elce, John S.

    PY - 1996/10/15

    Y1 - 1996/10/15

    N2 - 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.

    AB - 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.

    KW - Rats

    KW - Animals

    KW - Proline

    KW - Calcium

    KW - Hydrogen-Ion Concentration

    KW - Point Mutation

    KW - Calpain

    KW - Aspartic Acid

    KW - Substrate Specificity

    KW - Protein Conformation

    M3 - Article

    VL - 319

    SP - 535

    EP - 541

    JO - Biochemical Journal

    JF - Biochemical Journal

    SN - 0264-6021

    IS - (Pt2)

    ER -