Structure and Function of the Xenobiotic Substrate Binding Site of a Glutathione S-Transferase as Revealed by X-ray Crystallographic Analysis of Product Complexes with the Diastereomers of 9-(S-Glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene

Abstract

The three-dimensional structures of isoenzyme 3-3 of glutathione (GSH) transferase complexed with (9R,10R)- and (9S,10S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene [(9R,10R)-2 and (9S,10S)-2], which are the products of the addition of GSH to phenanthrene 9,10-oxide, have been determined at resolutions of 1.9 and 1.8 A, respectively. The structures indicate that the xenobiotic substrate binding site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, and L12 from domain I (the GSH binding domain) and I111, Y115, F208, and S209 in domain II of the protein. All of these residues are located in variable-sequence regions of the primary structure of class mu isoenzymes. Three of the eight residues (V9, I111, and S209) of isoenzyme 3-3 that are in direct van der Waals contact with the dihydrophenanthrenyl portion of the products are mutated (V9I, I111A, and S209A) in the related isoenzyme 4-4. These three residues are implicated in control of the stereoselectivity of the class mu isoenzymes. The hydroxyl group of Y115 is found to be hydrogen-bonded to the 10-hydroxyl group of (9S,10S)-2, a fact suggesting that this residue could act as an electrophile to stabilize the transition state for the addition of GSH to epoxides. The Y115F mutant isoenzyme 3-3 is about 100-fold less efficient than the native enzyme in catalyzing the addition of GSH to phenanthrene 9,10-oxide and about 50-fold less efficient in the Michael addition of GSH to 4-phenyl-3-buten-2-one. The side chain of Y115 is positioned so as to act as a general-acid catalytic group for two types of reactions that would benefit from electrophilic assistance. The results are consistent with the notion that domain II, which harbors most of the variability in primary structure, plays a crucial role in defining the substrate specificity of class mu isoenzymes.

Polymorphism of the glutathione transferase subunit 3 in Sprague–Dawley rats involves a reactive cysteine residue

“…We examined whether the two GST 3-3 forms, the lysine\serine form and the asparagine\cysteine form, differed in enzyme activity, because these regions, adjacent to the C-terminus, are thought to be responsible for binding of hydrophobic substrates [45]. However, we did not observe any marked variation in specific activity values with various substrates.…”

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

“…Motif I1 is located far from the active site of GST (Figs. 4, 5); recent structural studies did not implicate any of the residues in this motif in binding of either of the substrates (Garcia-Saez et al, 1994;Ji et al, 1994). Rather, these experiments have shown that, unlike the GSHbinding site, the electrophile-binding site is formed by variable segments of the GSTs.…”

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.…”