Arginine Kinase: A Crystallographic Investigation of Essential Substrate Structure

• Other Authors: Clark, Shawn A. (Shawn Adam) (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Chemistry
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-3596

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. === A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Fall Semester, 2006. === November 2, 2006. === L-Ornithine, Phosphagen, Kinase, Arginine, Crystallography, L-Citrulline, D-Arginine === Includes bibliographical references. === Michael Chapman, Professor Co-Directing Dissertation; Tim Logan, Professor Co-Directing Dissertation; Ross Ellington, Outside Committee Member; Al Stiegman, Committee Member.