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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055837-02
Application #
2883063
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1998-03-01
Project End
2003-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Florida State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
020520466
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
Bush, D Jeffrey; Kirillova, Olga; Clark, Shawn A et al. (2011) The structure of lombricine kinase: implications for phosphagen kinase conformational changes. J Biol Chem 286:9338-50
Gattis, James L; Ruben, Eliza; Fenley, Marcia O et al. (2004) The active site cysteine of arginine kinase: structural and functional analysis of partially active mutants. Biochemistry 43:8680-9
Azzi, Arezki; Clark, Shawn A; Ellington, W Ross et al. (2004) The role of phosphagen specificity loops in arginine kinase. Protein Sci 13:575-85
Yousef, Mohammad S; Clark, Shawn A; Pruett, Pamela K et al. (2003) Induced fit in guanidino kinases--comparison of substrate-free and transition state analog structures of arginine kinase. Protein Sci 12:103-11
Pruett, Pamela S; Azzi, Arezki; Clark, Shawn A et al. (2003) The putative catalytic bases have, at most, an accessory role in the mechanism of arginine kinase. J Biol Chem 278:26952-7
Yousef, Mohammad S; Fabiola, Felcy; Gattis, James L et al. (2002) Refinement of the arginine kinase transition-state analogue complex at 1.2 A resolution: mechanistic insights. Acta Crystallogr D Biol Crystallogr 58:2009-17
Canonaco, Fabrizio; Schlattner, Uwe; Pruett, Pamela S et al. (2002) Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool. J Biol Chem 277:31303-9