MAPPING THE SUBSTRATE-BINDING SITE OF A HUMAN CLASS MU GLUTATHIONE TRANSFERASE USING NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

The substrate-binding site of a human muscle class mu glutathione transferase has been characterized using high-resolution nuclear magnetic resonance spectroscopy. Isotopic labeling has been used to simplify one-dimensional proton NMR spectra of the Tyr and His residues in the enzyme and two-dimensional carbon-proton spectra of the Ala and Met residues in the enzyme. The resonance lines from 8 of the 12 Tyr residues have been assigned using site-directed mutagenesis. Replacement of Tyr7 with Phe reduced the activity of the enzyme 100-fold. The proximity of His, Tyr, Ala, and Met residues to the active site has been determined using a nitroxide-labeled substrate analogue. This substrate analogue binds with high affinity (K(eq) = 10(6) M-1) to the enzyme and is a competitive inhibitor. None of the His residues are within 17 angstrom of the active site. Three of the assigned Tyr residues are greater than 17 angstrom from the active site. Quantitative measurement of paramagnetic line broadening of five additional Tyr residues places them within 13-17 angstrom from the active site. Broadening of the Ala and Met resonance lines by the spin-labeled substrate indicates that three Ala residues are 9-16 angstrom from the nitroxide, three Met residues are less than 9 angstrom from the nitroxide, and two Met residues are 9-16 angstrom from the nitroxide. These data indicate that the active site region of this class mu glutathione transferase is in a location similar to that found for the class pi enzyme as described by Reinemer et al.