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

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

C2 - 8912692

SN - 0264-6021

VL - 319

SP - 535

EP - 541

JO - Biochemical Journal

JF - Biochemical Journal

IS - (Pt2)

ER -