Substrate polarization by residues in Δ5‐3‐ketosteroid isomerase probed by site‐directed mutagenesis and UV resonance Raman spectroscopy

Abstract

Δ⁵-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of Δ⁵-3-ketosteroids to Δ⁴-3-ketosteroids by the stereospecific transfer of the steroid 4β-proton to the 6β-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14₀ mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14₀ mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14₀ enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm⁻¹, respectively, on binding to Y14₀ KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14₀ enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in α-helical structure. The δ-helix content was estimated to be in the range of 0–25% (i.e., 10 ± 15%).