super(15)N NMR Relaxation Studies of Backbone Dynamics in Free and Steroid-Bound Delta super(5)-3-Ketosteroid Isomerase from Pseudomonas testosteroni

The backbone dynamics of Delta super(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by super(15)N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S super(2), tau sub(e), and R sub(ex)) and the overall rotational correlation time ( tau sub(m)). The rotational correlation times were 19.23 plus or minus 0.08 and 17.08 plus or minus 0.07 ns with the diffusion anisotropies (D sub( parallel )/D sub( perpendicular )) of 1.26 plus or minus 0.03 and 1.25 plus or minus 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S super(2)) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.