Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans， transforming Δ-androstene-3-17-dione into Δ-androstene-3-17-dione. GSTA3-3 catalyzes this reaction with ten-fold greater efficiency than GSTA1-1， its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable， subtle d... More
Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans， transforming Δ-androstene-3-17-dione into Δ-androstene-3-17-dione. GSTA3-3 catalyzes this reaction with ten-fold greater efficiency than GSTA1-1， its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable， subtle differences at the G-site of the enzymes account for GSTA3-3 having a ten-fold greater affinity for the substrate GSH. Y9F and R15L mutations， singly or together， have no effect on the structure and stability of GSTA3-3 (the same effect they have on GSTA1-1) despite the R15L mutation removing an interdomain salt-bridge at the active site. Hydrogen-deuterium exchange mass spectrometry also revealed that neither mutation had a significant effect on the conformational dynamics of GSTA3-3. The R15L and Y9F mutations are equally important to the specific activity of the steroid isomerase reaction; however， Arg15 is more important for lowering the pK of GSH. Lowering the pK of GSH being how GSTs catalyze their reactions. Additionally， there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ-androstene-3-17-dione and Δ-androstene-3-17-dione， indicating that Arg15 is a more important active-site residue than previously known.
