Why is creatine kinase a dimer? Evidence for cooperativity between the two subunits

Abstract

The dimeric chicken brain type isoenzyme of creatine kinase (BB-CK) was mutated by a C283S amino acid exchange in the catalytic site to produce a basically inactive dimer (B*B*-CK). The mutated enzyme showed a residual activity of about 4% compared to the wild-type, whereas substrate binding parameters were not altered. The inactivated dimer was hybridized with native dimeric muscle enzyme (MM-CK) to produce a partially inactivated MB*-CK heterodimeric hybrid and also to a his-tagged BB-CK (hBhB-CK) resulting in a partially inactive hBB*-CK homodimer. The generated hybrids were purified by chromatography. The V max and substrate binding parameters K m and K d were determined for both directions of the CK reaction and compared to the parameters of the wild-type enzymes (MM-, BB-, hBhB-, MB-CK). In the direction of ATP synthesis (reverse reaction), the MB*- and hBB*-CK hybrids showed a decrease of V max to 34% and 32%, respectively, compared to the unmodified wild-type isoform. The inactivation of a single subunit in MB*-CK led to an increase in the K d value resulting in an significant substrate synergism, not seen with the MB-CK wild-type enzyme. In the direction of phosphocreatine synthesis (forward reaction), the modified hybrids showed a decrease of V max to 50% of the wild-type enzymes and no significant alterations of the K m and K d parameters. These results strongly suggest an enzymatic cooperativity of the two subunits in the reverse reaction but independent catalytic function in the forward reaction.