Mutagenesis of two acidic active site residues in human muscle creatine kinase: Implications for the catalytic mechanism

Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanid ine substrate, creatine, by MgATP. Although several X-ray crystal structure s of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homol ogue, arginine kinase (AK), complexed with the transition-state analogue (a rginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu 225 and Glu314) within 2.8 Angstrom of the proposed transphosphorylation si te. These two residues are the putative catalytic groups that may promote n ucleophilic attack by the guanidine amino group on the gamma -phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kina ses, we have identified two homologous creatine kinase acidic amino acid re sidues (Glu232 and Asp326), and these were targeted for examination of thei r potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicat e that of these two residues the Glu232 is the likely catalytic residue whi le Asp326 likely performs a role in properly aligning substrates for cataly sis.