We propose to use the approaches of the bioorganic chemist as well as methods of the mechanistic enzymologist to probe the nature of the active site of creatine kinase, an enzyme that uses creatine, phosphocreatine, ADP and ATP as substrates. Isoenzymes of creatine kinase serve a dual role as objects for screening creatine, phosphocreatine, and nucleotide analogs of potentially great interest in other biological systems. They also serve as prototypal kinases, an increasingly important class of enzymes in biochemistry, the mechanism of action for none of which is well-understood. Our general approach involves development of novel inhibitors that are substrate analogs to probe the enzyme's active site, both to see which functional groups of the substrates are most vital to activity and to see what additional groups may be added without severe detrimental effect on activity. Such analogs may be novel substrates, competitive inhibitors or affinity labels. Various physical and biochemical methods, including kinetic studies, nuclear magnetic resonance studies, chemical modification and peptide-sequencing are proposed in the probing process. X-Ray crystallography will be used to determine the crystal structure of the enzyme. Computer graphics will be used in molecular modeling. Site-directed mutagenesis of the cloned gene will be used to help elucidate the possible roles of specific amino acid residues in the catalytic mechanism. Information gleaned from these studies may enable us to develop inhibitors of the various isoenzymes in their natural habitat with high selectivity so that, by differences, their physiological roles can be further elucidated. It may be possible to design methods of detecting one isoenzyme in the presence of others based on specificity differences. This could have clinical value in the detection of myocardial infarctions and other muscular disorders such as muscular dystrophy.
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