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 -GLUTATHIONYL)-10-HYDROXY-9,10-DIHYDROPHENANTHRENE

The three-dimensional structures of isoenzyme 3-3 of glutathione (GSH) transferase complexed with (9R,10R)- and -glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-2 and (9S,10S)-2], which are the prod ucts of the addition of GSH to phenanthrene 9,10-oxide, have been dete rmined at resolutions of 1.9 and 1.8 Angstrom, respectively. The struc tures indicate that the xenobiotic substrate binding site is a hydroph obic cavity defined by the side chains of Y6, W7, V9, and L12 from dom ain I (the GSH binding domain) and I111, Y115, F208, and S209 in domai n II of the protein. All of these residues are located in variable-seq uence regions of the primary structure of class mu isoenzymes. Three o f 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 isoen zyme 4-4. These three residues are implicated in control of the stereo selectivity 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 stab ilize the transition state for the addition of GSH to epoxides. The Y1 15F mutant isoenzyme 3-3 is about 100-fold less efficient than the nat ive enzyme in catalyzing the addition of GSH to phenanthrene 9,10-oxid e 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 a ct as a general-acid catalytic group for two types of reactions that w ould 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.