ULTRAVIOLET SPECTROSCOPIC EVIDENCE FOR DECREASED MOTION OF THE ACTIVE-SITE TYROSINE RESIDUE OF DELTA(5)-3-KETOSTEROID ISOMERASE BY STEROID-BINDING

Citation
Qj. Zhao et al., ULTRAVIOLET SPECTROSCOPIC EVIDENCE FOR DECREASED MOTION OF THE ACTIVE-SITE TYROSINE RESIDUE OF DELTA(5)-3-KETOSTEROID ISOMERASE BY STEROID-BINDING, Biochemistry, 34(19), 1995, pp. 6562-6572
Citations number
39
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
34
Issue
19
Year of publication
1995
Pages
6562 - 6572
Database
ISI
SICI code
0006-2960(1995)34:19<6562:USEFDM>2.0.ZU;2-V
Abstract
Delta(5)-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testost eroni catalyzes the highly efficient conversion of Delta(5)-3-ketoster oids to Delta(4)-3-ketosteroids by a stereoselective and intramolecula r transfer of the 4 beta-proton to the 6 beta-position. Tyr-14 is the critical general acid and Asp-38 is the general base involved in catal ysis. The UV absorption bandwidths of Tyr-14 were much narrower than t hose of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility. Further immobilization of this residue occurs upon steroid binding. Thus, 5 alpha-estrane-3,17-dione, an A-ring saturated steroid, induces significant narrowing of the tyr osine absorption bands (pi --> pi) of the main peak (279.5 nm) and th e shoulder (285.5 nm) of Tyr-14, with no significant changes in lambda (max). No effects of steroid binding were found on the absorption band widths of Tyr-55, Tyr-88, or the phenylalanine residues. The ratio of the absorbance (A(max)) at the absorption maximum (lambda(max)) to tha t at lambda(max) plus 4 nm (A(max+4)) was used as a measure of peak sh arpness. Specifically, the ratios of A(279.5)/A(283.5) (main peak) and A(285.5)/A(289.5) (shoulder) of Tyr-14 of the free enzyme at 25 degre es C were 1.25 and 1.89, respectively, and they increased to 1.41 and 2.70, respectively, in the complex. A more precise measurement of the band narrowing from 4.2 to 3.1 nm between the inflection points was ob tained from the derivative spectra. The absorption bands of free and s teroid-bound isomerase were narrowed significantly by lowering the tem perature and were broadened by denaturation, suggesting that the unusu al peak-sharpening effects induced by steroid binding arise from the r estricted motion of Tyr-lit, as well as from more directional hydrogen bonding resulting from the displacement of water molecules from the a ctive site and decreased flexibility of the protein. Larger enthalpy o f the sharpening effects was observed for the steroid-bound enzyme (-0 .527 +/- 0.016 kcal/mol) than for the free enzyme (-0.250 +/- 0.018 kc al/mol) by lowering the temperature, indicating that interactions of T yr-14 with its environment, which restrain its motion, are stronger in the steroid-bound enzyme than in the free enzyme. Hydrogen-bonding mo des of Tyr-14, mobility of the active site, and protein flexibility ar e the environmental factors determining the absorption bandwidths of t he critical Tyr-14 residue.