Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3

Abstract

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Δ5-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 × 105-fold decrease in kcat; the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in kcat/Km for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation kcat/Km was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. Ki values for Δ5-androstene-3,17-dione and Δ4-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pKa of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pKa of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of kcat/Km of wild-type GST A3-3 for the isomerase reaction displays two kinetic pKa values, 6.2 and 8.1. The basic limb of the pH dependence of kcat and kcat/Km disappears in the Y9F mutant. Therefore, the higher kinetic pKa reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.