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 guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 A of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the gamma-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.
Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
J S
JS
Cantwell
W R
WR
Novak
P F
PF
Wang
M J
MJ
McLeish
G L
GL
Kenyon
P C
PC
Babbitt
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003402:Creatine Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D052283:Creatine Kinase, MM Form; D003404:Creatinine / Q000031:analogs & derivatives / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D001224:Aspartic Acid; D018698:Glutamic Acid; C012260:cyclocreatine; D003404:Creatinine; D003402:Creatine Kinase; D052283:Creatine Kinase, MM Form