Preparation and properties of S-cyano derivatives of creatine kinase.
The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5'-dithio-bis(2-nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol-blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8 - 0.9-SH group per subunit. This mono-S-cyano derivative of creatine kinase was found 73% as active as the native unmodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted in the bis-S-cyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme.
D000818:Animals; D001665:Binding Sites; D003402:Creatine Kinase / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D011817:Rabbits; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D003486:Cyanides; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D003402:Creatine Kinase