The broad objectives are to understand the catalysis of cellular ATP energy buffering, and mechanisms of substrate specificity in metabolic enzymes, both at atomic resolution. Phosphagen (guanidino) kinases buffer ATP (temporally and/or spatially) in tissues with highly variable energy turnover, e.g. nerve and muscle. Detailed understanding of phosphagen kinase catalysis is important particularly in understanding how energy for muscle contraction is supplied with minimal change in ATP concentration, to understand the molecular physiology of heart and other tissues, and, more generally, in understanding enzyme catalysis of bimolecular reactions. Current knowledge of substrate specificity is based primarily on enzymes (e.g. proteases) with larger substrates and higher specificity than most metabolic enzymes. Phosphagen kinases will be developed as a paradigm for the mechanisms of specificity, taking advantage of their diverse array of substrates, yet high sequence conservation. Arginine kinase (AK), the """"""""primordial"""""""" phosphagen kinase, will be used as a model to understand human creatine kinase (CK) whose active site has eluded detailed characterization. The structure of a transition state analog (TSA) complex will be determined at 1.9 A and refined to at least 1.5 A resolution, using the results of preliminary work: a high yield expression system, high quality crystals, and phases from multiple isomorphous replacement, molecular replacement and averaging between two crystal forms. The substrate-induced conformational changes required to prevent non-productive ATP hydrolysis will be characterized using crystals of the apo-enzyme. Roles for active site residues in catalysis and specificity, and details of the catalytic mechanism will be determined. Roles proposed on the basis of observed atomic interactions, sequence conservation and kinetic data will be tested through site-directed mutagenesis, followed by kinetic and structural characterization. Kinetic parameters for wild-type and mutants with respect to various substrates and inhibitors (arginine, creatine etc.) will be correlated with the structures of corresponding enzyme complexes. Studies of specificity, starting with comparisons of small and intermediate substrates (creatine & arginine) will be extended to large substrates with studies of lombricine kinase (LK): first, through sequence determination and alignment, then kinetically and structurally following expression.
