title: Determination of the catalytic site of creatine kinase by site-directed mutagenesis.
abstract: Site-directed mutagenesis was used to alter the amino-acid residues at the presumed catalytic site Cys-283 and ATP binding site Asp-340 of human creatine kinase B cDNA. In addition, a highly conserved arginine residue, Arg-292, was also mutated. Transfection of 0.1 to 1 microgram of recombinant plasmid into COS cells produced increasing creatine kinase activity in the cell lysate. The expression of mutant Cys283-Tyr and Cys283-Ser resulted in complete abolition of homodimer BB isoform enzymatic activity without alteration of the capacity for dimerization. Expression of mutants Arg292-His, Arg292-Leu, and Arg292-Gln produced non-functional homodimers, whereas expression of mutant Arg292-Lys produced a homodimer with enzymatic activity that was 42% of the enzymatic activity of the wild type. Expression of the Asp340-Glu mutant creatine kinase did not alter enzyme activity as compared to the wild type. Following heterodimerization, there was inhibition of the normal subunit by the mutant subunit, for both the BB and the MB dimer. The results showed residues Cys-283 and Arg-292 are essential for enzyme catalysis. The best fit model for the dimer is one in which there is close apposition of the two catalytic sites. The interaction of the individual subunits during dimerization provides a molecular approach for dominant negative modulation of the creatine kinase isozyme system in future genetic manipulative experiments.
authors.affiliation: Methodist Hospital, Houston, TX.
authors.forename: L
authors.initials: L
authors.lastname: Lin
authors.forename: M B
authors.initials: MB
authors.lastname: Perryman
authors.forename: D
authors.initials: D
authors.lastname: Friedman
authors.forename: R
authors.initials: R
authors.lastname: Roberts
authors.forename: T S
authors.initials: TS
authors.lastname: Ma
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
chemical_list: D018076:DNA, Complementary; D003402:Creatine Kinase