title: 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.
authors.affiliation: Swiss Federal Institute of Technology, Institute of Cell Biology HPM F44, ETHZ-Hönggerberg, 8093, Zürich, Switzerland. horneman@cell.biol.ethz.ch
authors.forename: T
authors.initials: T
authors.lastname: Hornemann
authors.forename: D
authors.initials: D
authors.lastname: Rutishauser
authors.forename: T
authors.initials: T
authors.lastname: Wallimann
mesh_terms: D000818:Animals; D002645:Chickens; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D007700:Kinetics; D016297:Mutagenesis, Site-Directed
chemical_list: D003402:Creatine Kinase