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

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 Angstrom, 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.