Structure and Function of the Xenobiotic Substrate-Binding Site and Location of a Potential Non-Substrate-Binding Site in a Class π Glutathione S-Transferase^,

Abstract

Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

“…When the enzyme subunits are separated, Asp 98 from the opposite subunit is unavailable and the K m for GSH increases. In contrast, the site for CDNB is defined entirely by the side chains of residues within the same subunit [28]; thus separation of the subunits does not affect the binding by GST pi of this xenobiotic substrate. Furthermore, as indicated in the results from the effect of KBr on V max , the GST pi monomer is catalytically competent; in fact, the V max of the dimer is only 2.6 times that of the monomer.…”

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

“…A recent crystallographic study of the class pi GST [29] has shown that one molecule of N-(2-hydroxethyl)piperazine-N′-2-ethanesulphonic acid (Hepes) is bound in the hydrophobic cavity between the secondary structural elements β2 and A1. The binding interaction between BSP and human GST A1-1 was carried out in 0.025 MϪ0.4 M Hepes, 0.02% NaN 3, pH 6.5, to assess whether this buffer has any impact on BSP binding.…”

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

“…The catalytic diversity of mammalian cytosolic GSTs, which can exist as homo-or heterodimers, arises in part from the existence of at least eight distinct classes (named Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta) (1,4). The glutathione-binding site (G-site) and the catalytic mechanism of these enzymes have been the targets of many investigations involving chemical modification (5)(6)(7)(8), site-directed mutagenesis (9 -14), and x-ray crystallographic analysis (15)(16)(17)(18)(19)(20)(21). On the contrary, the electrophilic substrate-binding site (H-site) of GSTs is more complex and is class-specific.…”

“…Crystallographic studies have shown that the H-site is quite dissimilar among different classes of GSTs (15)(16)(17)(18)(19)(20)(21). For example, in GST M1-1, the H-site is a hydrophobic cavity, whereas in GST P1-1 (15), the H-site is half-hydrophobic and half-hydrophilic with functionally important water molecules (16). Only a few amino acid residues have been identified in the H-site of GSTs, and relatively little is known about the key determinants of xenobiotic substrate specificity.…”