Rate-limiting domain and loop motions in arginine kinase.

Arginine kinase (E.C. 2.7.3.3) is a member of the phosphagen kinase family of enzymes and catalyzes reversible transfer of a phosphoryl group between ATP and arginine. Found primarily in invertebrates, arginine kinase acts to buffer cellular levels of ATP in cells with high or variable demands for energy (1). In addition to its important physiological role, the phosphagen kinase family has been the focus of much research towards understanding bimolecular enzyme catalysis (1). Arginine kinase from the Atlantic horseshoe crab Limulus polyphemus, a 42 kDa monomer, is selected as a model system for investigating the link between enzyme dynamics and function, in part due to its amenability to both high resolution x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy (2–6). Steady-state enzyme kinetics have revealed the arginine kinase kinetic mechanism to be a rapid equilibrium, random order bimolecular-bimolecular reaction, with a turnover number (kcat) of approximately 135 s−1 at 25 °C (7–9). The enzyme has also been shown to undergo large conformational changes during the catalytic cycle. Initially, x-ray solution scattering experiments revealed a decrease in the radius of gyration of the enzyme associated with substrate binding (10). The locations and degree of these protein conformational changes were not evident, however, until x-ray crystal structures of the enzyme became available. Two crystal structures of Limulus polyphemus arginine kinase highlight the conformational changes associated with substrate binding. The first, in a substrate-free or “open” state, was initially solved at 2.4 A resolution and recently extended to 1.7 A (4, 11). The second structure, with a transition state analog bound to form a “closed” state, was solved at 1.2 A resolution and contained enzyme-bound MgATP, arginine, and nitrate, with the latter mimicking the phosphoryl group being transferred (5, 6). Overall, these structures established arginine kinase as a two-domain enzyme, with a primarily α-helical N-terminal domain (NTD) spanning residues 1–90 and a primarily β-sheet C-terminal domain (CTD) spanning residues 120–357. These two domains are joined via a 29 residue flexible linker. Comparison of these crystal structures shows a closing of the NTD and CTD along with folding of a set of loops over the active site (Figure 1). In general terms, the substrate associated conformational changes have been described as a hand making a grabbing motion (4–6, 11). A similar set of conformational changes have been observed in creatine kinase, an arginine kinase homologue (12–14). Figure 1 Conformational changes associated with substrate binding in arginine kinase. The stereo pair shows the substrate-free (opaque) and transition state analogue (transparent) crystal structures of arginine kinase. Sub-domains 1–5 are colored blue, ... The open and closed crystal structures of arginine kinase provide “snapshots” of the enzyme during the catalytic cycle. They give insight into the amplitudes of conformational changes but not the associated rates. Recently, NMR-based 15N spin relaxation experiments on substrate-free arginine kinase have highlighted the backbone dynamics intrinsic to the enzyme on a wide range of timescales (3). Lipari-Szabo analysis pinpointed loop L13, residues V308–V322, with intrinsic dynamics on the nanosecond timescale (15, 16). 15N transverse relaxation dispersion experiments indicated that approximately 12% of the protein was involved in micro- to millisecond timescale dynamics in the absence of substrate (17–19). Residues exhibiting these slow dynamics localized to four regions in the enzyme: (1) the interface and (2) hinge between the NTD and CTD; (3) loop L8, spanning residues I182-G209; and (4) several segments of the primary structure that together form the center of the CTD β-sheet (Figure 1). Interestingly, the rates of motion for the NTD and loop L8 were commensurate with the turnover rate, suggesting that one or both of these conformational changes might be rate-limiting upon turnover (3). The importance of protein motions for enzyme function has long been hypothesized; furthermore, there has been long-standing speculation that protein conformational changes could be rate limiting for some (20). It is only recently, however, that experimental techniques have been developed to probe conformational changes. Recent advances in NMR have allowed for the direct measurement of rates of protein motions at the residue level (21). This technique has allowed for determination of protein motions critical for turnover in systems such as triosephosphate isomerase, cyclophillin A, RNase A, and dihydrofolate reductase (21–24). The work presented here builds upon the previous work exploring the intrinsic dynamics of arginine kinase (3). To further test the hypothesis that protein conformational changes are rate-limiting on turnover, 15N transverse relaxation dispersion experiments along with enzyme kinetic measurements are performed across a temperature range of 15 to 30 °C (17–19). Conformational exchange rates (kex) and turnover rates (kcat) are obtained as a function of temperatures, thus allowing access to activation energies associated with intrinsic conformational changes and the entire catalytic cycle, respectively. The activation barriers of the NTD and loop L8 motions were found to be similar to that of the catalyzed reaction, thus providing further evidence that one or both of these conformational changes are rate-limiting upon arginine kinase turnover.