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. 2014 Nov 14;289(46):32243-32252.
doi: 10.1074/jbc.M114.601609. Epub 2014 Sep 23.

The isomerization of Δ5-androstene-3,17-dione by the human glutathione transferase A3-3 proceeds via a conjugated heteroannular diene intermediate

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The isomerization of Δ5-androstene-3,17-dione by the human glutathione transferase A3-3 proceeds via a conjugated heteroannular diene intermediate

Jonathan L Daka et al. J Biol Chem. .

Abstract

The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ(5)-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

Keywords: Androstene-3,17-dione; Enzyme Catalysis; Glutathione Transferase; Intermediate; Isotope Effect; Spectroscopy; Steroidogenesis; Thermodynamics; Transition State.

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Figures

FIGURE 1.
FIGURE 1.
Proposed reaction mechanisms for Δ5-AD isomerization by hGST A3-3. In Scheme I, adapted from Gu et al. (10), GSH abstracts the carbon 4 proton, and a water molecule acts as a hydrogen bond donor, stabilizing the negative charge on the dienolate intermediates (a and b panels). The keto form is regenerated by the transfer of negative charge via a conjugation system of π bonds with Tyr-9 acting as a proton shuttle (c). In Scheme II, adapted from Tars et al. (11), GSH abstracts a proton from carbon 4 while simultaneously transferring it to carbon 6. The keto group at carbon 3 is unaffected, and the reaction proceeds without the formation of the dienolate intermediate via a concerted single step mechanism.
FIGURE 2.
FIGURE 2.
The deprotonation of GSH expressed as a function of pH in the presence and absence of hGST A3-3. Nonlinear regression analysis of the data yielded a pKa value of 9.17 ± 0.04 for free GSH (●) and a pKa value of 6.31 ± 0.07 for the active site-bound GSH (○).
FIGURE 3.
FIGURE 3.
A, fluorescence emission spectra of equilenin as an indicator of the intermediate ionization state at the active site. Free equilenin (3.8 μm) is observed at pH 8.0 (red) and pH 11.0 (black) and in the presence of 9 μm enzyme (green). Equilenin was dissolved in 1% methanol, and excitation was set at 292 nm. B, the absorbance spectra of 19-nortestosterone (36.6 μm) observed in solution at pH 8.0 (red), in 10 m HCl (black), and in the presence of 19 μm enzyme (green).
FIGURE 4.
FIGURE 4.
A, the absorbance spectrum of 150 μm Δ5-AD in methanol. The isolated ethylenic bond absorbs maximally at 200 nm. B, the reaction progress curves of Δ5-AD followed at 238 nm (green), 256 nm (red), and 248 nm (black).The reactions were done with 1 μm enzyme and 9 μm substrate in 25 mm sodium phosphate, pH 8.0, at 20 °C.
FIGURE 5.
FIGURE 5.
The Arrhenius plots for the formation of product (Δ4-AD) followed at 248 nm yielded an Ea value of 13. 8 ± 0.5 kcal/mol (●) and the formation of the conjugated heteroannular diene intermediate followed at 238 nm yielded an Ea value of 12.9 ± 0.4 kcal/mol (○).
FIGURE 6.
FIGURE 6.
The fluorescence excitation (A) of equilenin and hGST A3-3 used to obtain a binding curve (B). The fluorescence data in A were collected for free equilenin (red), equilenin in the presence of hGST A3-3 (green), and free hGST A3-3 (black). A quenching effect is observed in the excitation spectra. The emission wavelength was set at 400 nm for A, and excitation was set at 325 nm for B. The data were fit to a three-parameter hyperbolic decay curve yielding a KD value of 3.93 ± 0.53 μm. Measurements were done in 1% methanol at pH 8.0 with 3 μm equilenin.
FIGURE 7.
FIGURE 7.
The proposed reaction pathway for the isomerization of Δ5-AD by hGST A3-3, were the steps are numbered 1–7 and the thermodynamic parameters are calculated per enzyme subunit. At step 1, GSH abstracts the carbon 4 proton; at step 2 a change in hybridization at carbon 4 to a higher energy p-orbital; at step 3, a transition state occurs that is nearly symmetrical but slightly favors the intermediate. At step 4 the structural realignments have advanced since the transition state, and the high energy p-orbital electrons are stabilized by the now fully developed conjugate system. At step 5 the transfer of a proton to carbon 6 is kinetically insignificant, resulting in an enforced concerted mechanism, and at step 6 a transfer of electrons from the p-orbital to the lower energy sp3 orbital resulting in step 7.

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