Glutathione transferase omega 1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones

Abstract

S-(Phenacyl)glutathione reductase (SPG-R) plays a significant role in the biotransformation of reactive alpha-haloketones to nontoxic acetophenones. Comparison of the apparent subunit size, amino acid composition, and catalysis of the reduction of S-(phenacyl)glutathiones indicated that a previously described rat SPG-R (Kitada, M., McLenithan, J. C., and Anders, M. W. (1985) J. Biol. Chem. 260, 11749-11754) is homologous to the omega-class glutathione transferase GSTO1-1. The available data show that the SPG-R reaction is catalyzed by GSTO1-1 and not by other GSTs, including the closely related GSTO2-2 isoenzyme. In the proposed reaction mechanism, the active-site cysteine residue of GSTO1-1 reacts with the S-(phenacyl)glutathione substrate to give an acetophenone and a mixed disulfide with the active-site cysteine; a second thiol substrate (e.g., glutathione or 2-mercaptoethanol) reacts with the active-site disulfide to regenerate the catalytically active enzyme and to form a mixed disulfide. A new spectrophotometric assay was developed that allows the rapid determination of SPG-R activity and specific measurement of GSTO1-1 in the presence of other GSTs. This is the first specific reaction attributed to GSTO1-1, and these results demonstrate the catalytic diversity of GSTO1-1, which, in addition to SPG-R activity, catalyzes the reduction of dehydroascorbate and monomethylarsonate(V) and also possesses thioltransferase and GST activity.

Figure 1

Reaction mechanism originally proposed for the glutathione-dependent, enzymatic reduction of 2,2′,4′-trichloroacetophenone (TCAP) to 2′,4′-dichloroacetophenone (DCAP). Modified from Ahktar (17) and Brundin et al. (20).

Figure 2

Structures of precursor α- and β-haloketones, S-(phenacyl)glutathiones, and S-(2-benzoylethyl)glutathione. 1a, 2-chloroacetophenone, X₁ = X₂ = H; 1b, S-(phenacyl)glutathione, X₁ = X₂ = H; 2a, 2,2′4′-trichloroacetophenone, X₁ = X₂ = Cl; 2b, S-(2′,4′-dichlorophenacyl)glutathione, X₁ = X₂ = Cl; 3a, 2-chloro-4′-fluoroacetophenone, X₁ = F, X₂ = H; 3b, S-(4′-fluorophenacyl)glutathione, X₁ = F, X₂ = H; 4a, 3-chloropropiophenone; 4b, S-(2-benzoylethyl)glutathione.

Figure 3

hGSTO1-1 catalyzed reduction of S-(phenacyl)glutathione 1b. S-(Phenacyl)glutathione 1b and 2-mercaptoethanol were incubated at 37 °C, and spectra were recorded at 1 min intervals, as described in Assay II (see Materials and Methods).

Figure 4

Proposed reaction mechanism for the hGSTO1-1-catalyzed reduction of S-(phenacyl)glutathiones to acetophenones. 1b, S-(phenacyl)glutathione, 2b, S-(2′,4′-dichlorophenacyl)glutathione, 3b, S-(4′-fluorophenacyl)glutathione, 5, keto-enol forms of product acetophenones, 6, product acetophenones.

Figure 5

Effect of pH on the SPG-R activity of hGSTO1-1. SPG-R activity was determined with S-(phenacyl)glutathione 1b and 2-mercaptoethanol as substrates in Assay II. Details of the buffers used have been described (21).

Figure 6

Expression of hGSTO1-1 in T47-D breast cancer cells. Panel A, Western blot of cell extracts probed with antiserum to human recombinant hGSTO1-1. Panel B, GST activity of cell extracts with CDNB as the substrate. Panel C, SPG-R activity of cell extracts determined with Assay II. 1, T47-D cells transfected with pcDNA3. 2, T47-D cells transfected with hGSTO1cDNA cloned in pcDNA3. Data are shown as means ± SD.