SUBUNIT-SELECTIVE CHEMICAL MODIFICATIONS OF CREATINE-KINASE - EVIDENCE FOR ASYMMETRICAL ASSOCIATION OF THE SUBUNITS

The 2 reactive thiol groups in the dimeric enzyme [rabbit muscle] creatine kinase (CK) react nonidentically with the cyanylating reagent 2-nitro-5-thiocyanobenzoic acid (NTCB). While in one subunit the thiol undergoes cyanylation, the other subunit thiol abnormally forms a mercaptonitrobenzoate (TNB) mixed disulfide. The resulting derivative, S-CN-S''-TNB-CK, is catalytically inactive. Cyanolysis of this derivative with KCN rapidly produces the dicyano enzyme S,S''-di-CN-CK, which possesses 75% of the original enzymic activity. The same active derivative is also formed by total cyanolysis of the inactive derivative S,S''-di-TNB-CK, produced by a previous reaction of the enzyme with 5,5''-dithiobis(2-nitrobenzoic acid) (DTNB). During the cyanolysis of S,S''-di-TNB-CK, one TNB group is displaced much more rapidly than the other. The regeneration of enzymic activity solely coincides with the faster of the 2 reactions. The 75%-active half-cyanolysis product, S-TNB-S''-CN-CK, is the isomer of the inactive S-CN-S''-TNB-CK produced by NTCB. These results suggest that the subunits of CK are asymmetrically associated. Difference spectrophotometry measurements showed that the inactive derivative S-CN-S''-TNB-CK is capable of forming the ternary complex E.cntdot.MgADP.cntdot.creatine at both of its subunits, but fails to form the quaternary transition-state analog (t.s.a) complex E.cntdot.MgADP.cntdot.NO3-.cntdot.creatine at either subunit. The 75% active S,S''-di-CN-CK is capable of forming the t.s.a. complex at each of the subunits. The single TNB blocking group in S-CN-S''-TNB-CK which prevents catalysis in the neighboring cyanylated subunit also eliminates the ability of that subunit to form the t.s.a. complex.