This project focuses on investigation of structure-function relationships in creatine kinase (C.K.), a phosphotransferase enzyme. The C.K.-catalyzed reaction acts as a chemical """"""""reservoir"""""""" to supply ATP to energy-requiring tissues such as muscle, heart, and brain. C.K. is important as a model system for understanding enzyme catalysis in general and in-line phosphoryl transfer mechanisms in particular. C.K. also serves as the primary clinical marker for myocardial infarct and is important in the diagnosis of many other pathologies ranging from muscular dystrophies to neurological damage. Research in recent years has revealed that C.K. may have a more important role in human physiology than was originally realized. The enzyme is now recognized to be ubiquitous, and its expression is regulated developmentally and by a range of hormones in a tissue-specific manner. Within the human cell, two cytosolic and two mitochondrial isoenzymes interact with different cellular structures, and evidence suggests that this intracellular localization has profound functional consequences for cellular energy balance. Primary sequences for creatine kinases from a range of species are available, and the applicants have recently found a homolog in Bacillus subtilis, extending the range of this unique class of enzymes into regions of the animal kingdom that have never been heretofore suspected. In this proposal, the investigators plan to focus our classical structure- function studies to answer remaining important questions about C.K. These include determination of the structural location and roles of active site residues in chemical catalysis and completion of the first x-ray crystallographic structure for this class of enzymes. They have also taken advantage of the explosion of information available from DNA sequencing projects that have produced 43 primary structures of C.K.'s and other guanidino kinase homologs to launch comparative studies among the isoenzymes. To enhance their capabilities for this aspect of the project, they have developed high efficiency bacterial expression and purification procedures and obtained clones for all of the human C.K. isoenzymes. This approach will provide insights that cannot be obtained from the most highly focused studies on a single isoenzyme. Because this enzyme family is so similar in structure, homology analysis of these 43 sequences provides clues about structural determinants of intracellular localization, antibody binding, and dimer/octamer formation. The resulting hypotheses can be tested by making chimeric forms of the isoenzymes in which a given structural region of one isoenzyme can be replaced with the analogous structural region from another. This will allow the investigators to isolate specific structural components and determine their contributions to function with minimal disturbance to the overall scaffold. The applicants believe that this is a novel strategy with the potential of contributing general insights into enzyme architecture associated with the roles of variable surface loops and oligomer formation as well.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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University of Michigan Ann Arbor
Schools of Pharmacy
Ann Arbor
United States
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