Robertson Gary J, Stoychev Stoyan H, Sayed Yasien, Achilonu Ikechukwu, Dirr Heini W
Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
CSIR Biosciences, Pretoria, South Africa.
Biophys Chem. 2017 May;224:40-48. doi: 10.1016/j.bpc.2017.02.004. Epub 2017 Mar 1.
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.
谷胱甘肽S-转移酶A3-3是人类已知的催化效率最高的类固醇异构酶，可将Δ-雄烯-3-17-二酮转化为Δ-雄烯-3-17-二酮。GSTA3-3催化该反应的效率比其在谷胱甘肽S-转移酶α类中最接近的竞争者GSTA1-1高十倍。为了研究α类谷胱甘肽S-转移酶之间的差异并更好地阐明GSTA3-3的机制，研究了Tyr9和Arg15的作用。Tyr9是α类谷胱甘肽S-转移酶的主要催化残基，有人提出Arg15对GSTA3-3的催化很重要，但以前从未进行过实验研究。虽然α类酶的结构和稳定性高度可比，但酶的G位点的细微差异导致GSTA3-3对底物谷胱甘肽的亲和力高十倍。Y9F和R15L突变单独或一起对GSTA3-3的结构和稳定性没有影响（它们对GSTA1-1也有相同的影响），尽管R15L突变消除了活性位点的域间盐桥。氢-氘交换质谱也表明，这两种突变对GSTA3-3的构象动力学都没有显著影响。R15L和Y9F突变对类固醇异构酶反应的比活性同样重要；然而，Arg15对于降低谷胱甘肽的pK更重要。降低谷胱甘肽的pK是谷胱甘肽S-转移酶催化其反应的方式。此外，有证据表明Arg15对于使GSTA3-3区分Δ-雄烯-3-17-二酮和Δ-雄烯-3-17-二酮是不可或缺的，这表明Arg15是一个比以前所知更重要的活性位点残基。
