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

Abstract

Δ 5‐3‐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%). Copyright © 1992 The Protein Society