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 (Keq = 10(6) M-1) to the enzyme and is a competitive inhibitor. None of the His residues are within 17 A of the active site. Three of the assigned Tyr residues are greater than 17 A from the active site. Quantitative measurement of paramagnetic line broadening of five additional Tyr residues places them within 13-17 A 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 A from the nitroxide, three Met residues are less than 9 A from the nitroxide, and two Met residues are 9-16 A from the nitroxide.(ABSTRACT TRUNCATED AT 250 WORDS)
Penington
C J
CJ
Department of Biochemistry, University of Virginia, Charlottesville 22908.
Rule
G S
GS
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003497:Cyclic N-Oxides; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011487:Protein Conformation; D013113:Spin Labels
D003497:Cyclic N-Oxides; D013113:Spin Labels; C029910:3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl; D005982:Glutathione Transferase; D005978:Glutathione