title: The isomerization of Δ5-androstene-3,17-dione by the human glutathione transferase A3-3 proceeds via a conjugated heteroannular diene intermediate.
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.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Daka
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa. Electronic address: heinrich.dirr@wits.ac.za.
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D000735:Androstenedione / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D009277:Nandrolone / Q000737:chemistry; D011485:Protein Binding; D011522:Protons; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds; D013816:Thermodynamics; D014466:Ultraviolet Rays
chemical_list: D007554:Isotopes; D011522:Protons; D013438:Sulfhydryl Compounds; D000735:Androstenedione; D009277:Nandrolone; D005982:Glutathione Transferase; C495975:glutathione S-transferase A3-3, human
keywords: Androstene-3,17-dione; Enzyme Catalysis; Glutathione Transferase; Intermediate; Isotope Effect; Spectroscopy; Steroidogenesis; Thermodynamics; Transition State