Key result
Mutating V325 to glutamate in human muscle creatine kinase drives a ~100-fold preference for glycocyamine.
Why the study?
There has been debate over the specific roles of flexible loops in substrate specificity and catalysis in creatine kinase and related phosphagen kinases.
This study demonstrates that residue V325 is critical for determining the substrate specificity of human muscle creatine kinase.
Cautions against clinical translation; extends mechanistic insight into creatine kinase substrate specificity for further study.
Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.
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Novak et al. (2004) studied this question. Mutagenesis of V325 (V325 to glutamate or alanine) was evaluated on Substrate specificity (>100-fold preference). Mutation of V325 to glutamate in human muscle creatine kinase resulted in a more than 100-fold preference for glycocyamine, altering the enzyme's substrate specificity.
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