Type of Document	Dissertation
Author	Clark, Shawn Adam
Author's Email Address	sclark2@bidmc.harvard.edu
URN	etd-11102006-131514
Title	Arginine Kinase; A Crystallographic Investigation of Essential Substrate Structure
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Michael Chapman Committee Co-Chair Tim Logan Committee Co-Chair Albert Stiegman Committee Member Ross W. Ellington Committee Member Tim Cross Committee Member
Keywords	L-Ornithine Phosphagen Kinase Arginine Crystallography L-Citrulline D-Arginine
Date of Defense	2006-11-02
Availability	unrestricted
Abstract Phosphagen kinases are a family of enzymes that play a role in high-energy cells by buffering the ATP concentration. Arginine kinase (AK) catalyzes the magnesium-dependent reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP. Previous investigations of arginine kinase have suggested that precise substrate alignment may play an important role in this bimolecular reaction and that phosphagen specificity is mediated by an induced fit mechanism. In order to uncover the mechanism of catalysis we present the crystallographic structures of arginine kinase with four arginine homologues: L-imino-ethyl-ornithine (ILO), L-citrulline (CIT), L-ornithine (ORN), and D-arginine (DARG) replacing the cognate arginine substrate. Each homologue was co-crystallized with MgADP- and nitrate as in the transition state analog complex (TSAC). The models were refined at 2.4Å, 2.0Å, 2.0Å and 2.8Å with Rfree values of 23%, 25%, 24% and 23%, respectively. These structures were used to study the basis of substrate specificity and the role of substrate alignment in catalysis. The non-cognate ligands bind and induce the substrate-bound enzyme conformation but are inactive. These data show that although AK is highly specific a number of different phosphagen mimics can bind, but are non-reactive. Comparison of these structures indicates that substrate specificity might be mediated by a mechanism that allows only the cognate substrate to be held rigidly in the proper orientation for efficient catalysis. These data also suggest that substrate alignment may play, at least, a role in catalysis. Here it is suggested that the induced fit movements of the enzyme most likely do not play a role in substrate discrimination but do play a role in substrate alignment. The structures also show that the guanidinium and α-amine moieties contribute little to substrate binding relative to the carboxylate, which appears to be key to substrate binding.
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