Rate-limiting domain and loop motions in arginine kinase

Abstract

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and arginine. It is the arthropod homologue of creatine kinase, buffering cellular ATP levels. Crystal structures of arginine kinase, in substrate-free and substrate-bound forms, have revealed large conformational changes associated with the catalytic cycle. Recent nuclear magnetic resonance identified movements of the N-terminal domain and a loop comprising residues I182--G209 with conformational exchange rates in the substrate-free enzyme similar to the turnover rate. Here, to understand whether these motions might be rate-limiting, we determined activation barriers for both the intrinsic dynamics and enzyme turnover using measurements over a temperature range of 15-30 °C. (15)N transverse relaxation dispersion yields activation barriers of 46 ± 8 and 34 ± 12 kJ/mol for the N-terminal domain and I182--G209 loop, respectively. An activation barrier of 34 ± 13 kJ/mol was obtained for enzyme turnover from steady-state kinetics. The similarity between the activation barriers is indeed consistent with turnover being limited by backbone conformational dynamics and pinpoints the locations of potentially rate-limiting motions.

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, red, green, cyan, and orange, respectively; flexible residues which are not a member of any quasi-rigid sub-domain are grey. Axes of rotation between sub-domains are shown with arrows. Residues previously shown by NMR relaxation dispersion experiments to be undergoing μs-ms timescale dynamics are shown in thicker trace (3).

Figure 2

(A) 2D [¹⁵N, ¹H] TROSY of arginine kinase recorded at 800 MHz at 15 °C (blue), 20 °C (cyan), 25 °C (green), and 30 °C (28). The boxed our region is expanded in the inset. (B) Collectively fit ¹⁵N transverse relaxation dispersion curves for T206 (left) and N137 (right), representative of loop L8 and the NTD-CTD interface, respectively. L31, a motionless control, is also shown. Data shown were collected at ¹⁵N Larmor frequency of 81.04 MHz at 15 °C (blue), 20 °C (cyan), 25 °C (green), and 30 °C (28). Errors are estimated from duplicate measurements of R₂^eff.

Figure 3

Arrhenius plots of k_cat (circles, solid line), collective k_ex for loop L8 (triangles, dotted line) and the NTD-CTD interface (squares, dashed line), and k_close for loop L8 (diamonds, dashed-and-dotted line). Errors are obtained from least-squares fitting (see Materials and Methods). The similarity of the slopes is indicative of similar activation barriers. Data shown here are for illustrative purposes; activation barriers were determined from least-squares fitting of the Arrhenius equation.

Figure 4

Rate-limiting conformational changes in arginine kinase. Shown is a ribbon diagram of the substrate-free crystal structure of arginine kinase, with N- and C-terminal domains labeled (39). Residues exhibiting chemical exchange, and used to determine activation barriers of intrinsic backbone dynamics, lie in two regions of the enzyme: loop L8 (I182-G209, red) and the interface between the N- and C-terminal domains, denoted by a dotted line (blue). The activation barriers for these two intrinsic motions are similar to that of enzyme turnover (34 ± 13 kJ/mol), indicating that one or both of these motions are likely involved in the rate-limiting step of catalytic turnover.