Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain.
In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-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 Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme.
Department of Biochemistry, Queen's University, Kingston, ON, Canada.
J S
JS
Arthur
J S
JS
Elce
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017354:Point Mutation; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity
D001224:Aspartic Acid; D011392:Proline; D002154:Calpain; D002118:Calcium