RUI: Comparative Structure/Function Analysis of Phosphagen Kinases

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Phosphagen kinases (PKs) are highly conserved enzymes found in all animals examined to date. They play a key role in energy homeostasis by maintaining stable ATP concentrations during times of high activity. Creatine kinase (CK) is the exclusive PK found in vertebrates, whereas arginine kinase (AK) is found in invertebrates. Unlike CK, the native state of AK is monomeric, except in a few cases. The reasons for this difference in quaternary structure is currently unknown, but may be based in differing requirements of physiological function or stability. The overall goal of this research is to utilize a comparative biochemical approach to extend current understanding of how structure determines function in PKs. These projects specifically focus on the interplay between substrate binding, inter-subunit cooperativity and bimolecular reaction mechanisms in PKs. PKs offer some unique advantages to the study of these issues given that they have evolved along different tracks, yet still perform the same basic physiological function. The specific goals of this research are to 1) investigate the amino acid determinants of inter-subunit communication (negative cooperativity) observed in rabbit muscle CK; 2) determine the minimal set of residues sufficient to generate a functional homodimeric form of horseshoe crab AK; 3) determine the structure and function of novel, putative PKs from Gram positive bacteria that appear to be missing the structural components necessary for guanidino-substrate specificity; 4) determine the structure and function of five, novel, putative AKs from Caenorhabditis elegans as a means of establishing a foundation for future experiments for placing these studies within a physiological context. Completion of these goals will provide an understanding of how the differences in primary structure between AK and CK give rise to the observed differences in quaternary structure, subunit cooperativity, substrate specificity and catalysis of phosphate transfer within this important and widespread protein family. Broad Impact: The College of Wooster has a national reputation for its Independent Study program. Excellent scientific training in modern biochemical and molecular biology techniques for a significant number of young potential scientists will be enhanced by this research. In addition, these projects serve as a model of collaborative research for undergraduate students in biology, chemistry and biochemistry / molecular biology.