title: Mechanism of the glutathione transferase-catalyzed conversion of antitumor 2-crotonyloxymethyl-2-cycloalkenones to GSH adducts.
abstract: Human glutathione (GSH) transferase (hGSTP1-1) processes with similar kinetic efficiencies the antitumor agents 2-crotonyloxymethyl-2-cyclohexenone (COMC-6), 2-crotonyloxymethyl-2-cycloheptenone (COMC-7), and 2-crotonyloxymethyl-2-cyclopentenone (COMC-5) to 2-glutathionylmethyl-2-cyclohexenone, 2-glutathionylmethyl-3-glutathionyl-2-cycloheptenone, and 2-glutathionylmethyl-2-cyclopentenone, respectively. This process likely involves initial enzyme-catalyzed Michael addition of GSH to the COMC derivative to give a glutathionylated enol(ate), which undergoes nonstereospecific ketonization, either while bound to the active site or free in solution, to a glutathionylated exocyclic enone. Free in solution, GSH reacts at the exomethylene carbon of the exocyclic enone, displacing the first GSH to give the final product. This mechanism is supported by the observation of multiphasic kinetics in the presence of high concentrations of hGSTP1-1 and the ability to trap kinetically competent exocyclic enones in aqueous acid using COMC-6 and COMC-7 as substrates. That the exocyclic enone is formed by nonstereospecific ketonization of an enol(ate) species is indicated by the observation that COMC-6 (chirally labeled with deuterium at the exomethylene carbon) gives stereorandomly labeled exocyclic enone. The isozymes hGSTP1-1, hGSTA1-1, hGSTA4-4, and hGSTM2-2 catalyze the conversion of COMC-6 to final product with similar efficiencies (K(m) = 0.08-0.34 mM, k(cat) = 1.5-6.1 s(-)(1)); no activity was detected with the rat rGSTT2-2 isozyme. Molecular docking studies indicate that in hGSTP1-1, the hydroxyl group of Tyr108 might serve as a general acid catalyst during substrate turnover. The possible significance of these observations with respect to the metabolism of COMC derivatives in multidrug resistant tumors is discussed.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA. hamilton@umbc.edu
authors.forename: Diana S
authors.initials: DS
authors.lastname: Hamilton
authors.forename: Xiyun
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhebo
authors.initials: Z
authors.lastname: Ding
authors.forename: Ina
authors.initials: I
authors.lastname: Hubatsch
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
authors.forename: K N
authors.initials: KN
authors.lastname: Houk
authors.forename: Bruce
authors.initials: B
authors.lastname: Ganem
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Creighton
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D003512:Cyclohexanones / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000970:Antineoplastic Agents; D003512:Cyclohexanones; D007527:Isoenzymes; C073431:2-crotonyloxymethyl-2-cyclohexenone; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D005978:Glutathione