Steady State Dynamics of Enzyme Catalytic Action in Solution

Abstract

Wide-angle X-ray scattering (WAXS) and neutron spin-echo (NSE) spectroscopy were used to study of the dynamics of adenylate kinase (Adk) as it undergoes steady state catalytic cycling. Analysis of the WAXS data indicates that it cannot be completely explained as a combination of open and closed states and suggests that ADP-bound Adk in solution mostly populates a closed conformation as demonstrated in earlier NMR studies [1,2,3]. The presence of other intermediates must be hypothesized to explain the observed data. Correlated internal dynamics on ps-ns timescales for the protein during active cycling appear to be slower than comparable dynamics in the open and closed states at certain length scales and faster at others. Our NSE results on active enzyme catalysis are consistent with the picture of energetic counterweight balancing on substrate binding. The enzyme appears to shift mobile regions during active catalysis for subsequent substrate release [4]. These methods will help define functional motions in proteins. Supported by DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494)[1] Henzler-Wildman, K.A., V. Thai, M. Le1, M. Ott, M. Wolf-Watz, T. Fenn, E. Pozharski, M. A. Wilson, G. A. Petsko, M. Karplus, C. G. Hubner, and D. Kern (2007b) Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838-844.[2] Jorgen Aden and Magnus Wolf-Watz, (2007) NMR Identification of Transient Complexes Critical to Adenylate Kinase Catalysis J. AM. CHEM. SOC., 129, 14003-14012[3] M. D. Daily, L. Makowski, G. N. Phillips, and Q. Cui, (2012) Large-scale motions in the adenylate kinase solution ensemble: Coarse-grained simulations and comparison with solution X-ray scattering, Chem. Phys. 396, 84-91.[4] Muller, C.W., Schlauderer, G.J., Reinstein, J. & Schulz, G.E. (1996). Adenylate kinase motions during catalysis, an energetic counterweight balancing substrate binding. Structure 4, 147-156.