Tyrosine 115 participates both in chemical and physical steps of the catalytic mechanism of a glutathione S-transferase.

The participation of the hydroxyl group of tyrosine 115 in the catalytic mechanism of isoenzyme 3-3 of rat glutathione (GSH) S-transferase is implicated by x-ray crystallographic analysis of a product complex and confirmed by comparison of the catalytic properties of the native enzyme and the Y115F mutant. Tyrosine 115 is located in domain II of the protein (the xenobiotic substrate binding domain) and is the first residue in this domain to be shown to play a direct role in catalysis. The 1.8-A structure of isoenzyme 3-3 in complex with (9S,10S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene, one of the diastereomeric products of the reaction of GSH with phenanthrene 9,10-oxide, indicates that the hydroxyl group of Tyr115 is within hydrogen-bonding distance of the 10-hydroxyl group of the bound product and, by implication, is proximal to the oxirane oxygen of the substrate in the Michaelis complex. Site-specific replacement of Tyr115 with phenylalanine has profoundly different effects on catalysis depending on the type of reaction and whether the rate-limiting step in catalysis is a chemical step or a physical step. Stopped flow measurements of the rate constants for product release and viscosity effects on the steady-state kinetics establish that the rate-limiting step in catalysis with phenanthrene 9,10-oxide (kcat = 0.4 s-1) is probably a chemical one, whereas the physical step of product dissociation (koff) is rate-limiting in the reaction of 1-chloro-2,4-dinitrobenzene (kcat = 20 s-1). The Y115F mutant is severely impaired in catalyzing the addition of GSH to phenanthrene 9,10-oxide (kcat = 0.0044 s-1), evidence that the -OH of Tyr115 provides electrophilic assistance in the epoxide ring opening. In contrast, the Y115F mutant is a better catalyst toward 1-chloro-2,4-dinitrobenzene (kcat = 72 s-1) than is the native enzyme. The enhanced rates of product release in the mutant are ascribed to the loss of hydrogen bonds between the -OH of Tyr115 and the side chain -OH and main chain NH of serine 209, interactions that block the channel to the active site or inhibit the segmental motion of the protein.