The effects of mutating Tyr9 and Arg15 on the structure, stability, conformational dynamics and mechanism of GSTA3-3.
Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans, transforming Δ5-androstene-3-17-dione into Δ4-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 pKaof GSH. Lowering the pKaof GSH being how GSTs catalyze their reactions. Additionally, there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ5-androstene-3-17-dione and Δ4-androstene-3-17-dione, indicating that Arg15 is a more important active-site residue than previously known.
Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Gary J
GJ
Robertson
CSIR Biosciences, Pretoria, South Africa.
Stoyan H
SH
Stoychev
Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Yasien
Y
Sayed
Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Ikechukwu
I
Achilonu
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.
Heini W
HW
Dirr
D001120:Arginine / Q000235:genetics*; D002384:Catalysis; D020134:Catalytic Domain; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055550:Protein Stability; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics*
D014443:Tyrosine; D001120:Arginine; D005982:Glutathione Transferase; C495975:glutathione S-transferase A3-3, human; D005978:Glutathione
GSTA3-3; HDX-MS; ITC; Mechanism; Stability; Steroid isomerase