Arginine kinase reversibly catalyzes phosphoryl transfer between ATP and arginine, thus providing a mechanism for buffering ATP levels in cells with high or variable energy requirements. X-ray crystal structures of a substrate-free and transition state analog form of arginine kinase suggest large conformational changes upon substrate binding. Steady state enzyme kinetics show that arginine kinase follows a random, bimolecular bimolecular kinetic mechanism with a turnover rate of ~135 sec¬¬-1. While the crystal structures have provided a wealth of information about the conformational changes of arginine kinase, they provide little to no data on dynamics. Crystal structures provide static snapshots at endpoints of rather complex equilibria. The link between enzyme dynamics and function is increasingly apparent but still remains relatively unexplored. Recently developed NMR techniques which probe dynamics on the micro- to millisecond timescale have provided insight into connection between dynamics and catalysis in a number of systems. The work presented in this dissertation is an NMR-based investigation into the dynamics or arginine kinase. Expression and purification of arginine kinase enriched with 15N, 13C, and 2H, a requirement for the NMR experiments, was achieved. Another prerequisite, resonance assignment, was accomplished using a standard suite of triple resonance NMR experiments and urea-induced unfolding and refolding to allow for back-exchange of amide deuterons in the core with solvent protons. Backbone amide resonances were assigned for 329 of 344 assignable residues. At the time, arginine kinase was one of the five largest monomeric units to be assigned. Using 15N transverse relaxation dispersion experiments, the dynamics of substrate-free arginine kinase were probed. These experiments implicate a number of residues, which cluster in four regions of the enzyme, in slow micro- to millisecond timescale dynamics. Most interesting is the loop spanning residues I182-G209, which the crystal structures show undergoes a large conformational change to interact with substrate nucleotide. The rate of exchange for this loop was found to be approximately 800 sec-1, on the same order as turnover, indicating that the motion associated with this loop may be a rate-limiting step upon catalysis. Furthermore, the changes associated with binding of substrates have been probed by substrate titrations in conjunction with 2D [15N, 1H]-TROSY spectroscopy. These experiments, which segregate the conformational changes seen in the crystal structures into those induced by binding of individual substrates, show that phosphagen and nucleotide binding elicits relatively independent changes in the N-terminal and C-terminal domains, respectively. The loop spanning residues I182-C201, however, appears to be affected by both substrates. Interestingly, this is the same loop relaxation dispersion experiments implicate in slow dynamics. As a bimolecular enzyme representative of a large enzyme class, the transferases, the amenability of arginine kinase to both x-ray crystallography and NMR make it a unique model system for understanding the connections between dynamics and function. The work described here outlines the potentially rate limiting intrinsic dynamics of arginine kinase and changes induced by substrate binding. These results highlight the importance of dynamics and reflect the growing view that enzymes have evolved both structure and dynamics simultaneously. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial
fulfillment of the requirements for the degree of Doctor of Philosophy. / Degree Awarded: Spring Semester, 2008. / Date of Defense: December 13, 2007. / Enzyme Dynamics, Relaxation Dispersion, Conformational Change, Arginine Kinase / Includes bibliographical references. / Michael S. Chapman, Professor Co-Directing Dissertation; Timothy M. Logan, Professor Co-Directing Dissertation; W. Ross Ellington, Outside Committee Member; John G. Dorsey, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_168966 |
| Contributors | Davulcu, Omar (authoraut), Chapman, Michael S. (professor co-directing dissertation), Logan, Timothy M. (professor co-directing dissertation), Ellington, W. Ross (outside committee member), Dorsey, John G. (committee member), Department of Chemistry and Biochemistry (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
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