Solvation Response along the Reaction Coordinate in the Active Site of Ketosteroid Isomerase

摘要

A light-activated reaction analog has been developed to mimic the catalytic reaction cycle of Δ~5-3-ketosteroid isomerase to probe the functionally relevant protein solvation response to the catalytic charge transfer. Δ~5-3-ketosteroid isomerase from Pseudomonas putida catalyzes a C-H bond cleavage and formation through an enolate intermediate. Conversion of the ketone substrate to the enolate intermediate is simulated by a photoacid bound to the active site oxyanion hole. In the ground state, the photoacid electrostatically resembles the enolate intermediate while the low pK_a excited state resembles the ketone starting material. Time-resolved fluorescence experiments with photoacids coumarin 183 and equilenin show the active site of Δ~5-3-ketosteroid isomerase to be largely unperturbed by the light-activated reaction. The small solvation response for the photoacid at the active site as compared with a simple solvent suggests the active site does not significantly change its electrostatic environment during the catalytic cycle. Instead, the reaction takes place in an electrostatically preorganized environment.