Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states

TY - JOUR

T1 - Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states

AU - Jensen, Kristine Steen

AU - Winther, Jakob R

AU - Teilum, Kaare

PY - 2011/3/9

Y1 - 2011/3/9

N2 - Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover with a combination of NMR magnetization transfer, R(2) relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically tuned to exchange on the millisecond time scale. The conformational changes of the protein backbone during catalysis were followed by (15)N nuclear spin relaxation dispersion experiments. A conformational transition that is well described by a two-state process with an exchange rate corresponding to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions on the time scale of catalytic turnover were not observed for the enzyme in the resting states, implying that alternative conformers do not accumulate to significant concentrations. These results infer that the turnover rate in glutaredoxin is governed by formation of a productive enzyme-substrate encounter complex, and that catalysis proceeds by an induced fit mechanism rather than by conformer selection driven by intrinsic conformational dynamics.

AB - Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover with a combination of NMR magnetization transfer, R(2) relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically tuned to exchange on the millisecond time scale. The conformational changes of the protein backbone during catalysis were followed by (15)N nuclear spin relaxation dispersion experiments. A conformational transition that is well described by a two-state process with an exchange rate corresponding to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions on the time scale of catalytic turnover were not observed for the enzyme in the resting states, implying that alternative conformers do not accumulate to significant concentrations. These results infer that the turnover rate in glutaredoxin is governed by formation of a productive enzyme-substrate encounter complex, and that catalysis proceeds by an induced fit mechanism rather than by conformer selection driven by intrinsic conformational dynamics.

KW - Catalysis

KW - Glutaredoxins

KW - Glutathione

KW - Humans

KW - Molecular Dynamics Simulation

KW - Nuclear Magnetic Resonance, Biomolecular

KW - Protein Binding

KW - Protein Conformation

U2 - 10.1021/ja1096539

DO - 10.1021/ja1096539

M3 - Journal article

C2 - 21323311

SN - 0002-7863

VL - 133

SP - 3034

EP - 3042

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 9

ER -