15N NMR Relaxation Studies of Backbone Motion of the catalytic Residues in Free and Steroid-bound Δ5-3-Ketosteroid Isomerase

Abstract

Backbone dynamics of the catalytic residues in free and steroid-bound Δ5-3- ketosteroid isomerase from Pseudomonas testosteroni has been examined by 15N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters ( , , and ). Tyr-14 and Asp-99 exhibit enhanced high-frequency (pico- to nanosecond) internal motions in the free enzyme, which are restricted upon ligand binding, while Asp-38 experiences severe restriction of the internal motions in the free enzyme, suggesting that Tyr-14 and Asp-99 are more actively involved in the ligand binding than Asp-38. The results also indicate that the H-bond network in the catalytic cavity might be slightly strengthened upon ligand binding, which may have some implications on the enzyme mechanism.