title: Unusual reactivity of Tyr-7 of GSH transferase P1-1.
abstract: Reaction of human GSH transferase P1-1 (GSTP1-1) with diethylpyrocarbonate (DEPC) at pH 7.0 and 4 degrees C resulted in covalent modification of an equivalent of one histidine and one tyrosine residue per subunit, with loss of activity. Sequence analysis showed that His-71 and Tyr-7 were modified. Reference to the three-dimensional structure of GSTP1-1 [Reinemer, Dirr, Ladenstein, Huber, Lo Bello, Frederici and Parker (1992) J. Mol. Biol. 227, 214-226] shows that the modification of Tyr-7 is most likely to affect enzyme activity. Kinetic analysis of the DEPC modification of Tyr-7 in GSTP1-1 gave a k2 approx. 150 times that of a peptide comprising residues 1-11 of GSTP1-1. The reaction of Tyr-7 of GSTP1-1 with DEPC was poorly inhibited by 1 mM GSH (14%) or 10 microM S-hexylglutathione (18%). DEPC treatment of the enzyme altered the absorbance at 290 nm in second-derivative spectra, suggesting that a significant amount of tyrosinate ion occurs in the enzyme. GSH, however, did not significantly alter the A290. The data provide the first evidence of unusual chemical reactivity of Tyr-7 and are consistent with its proposed role as a proton acceptor during catalysis.
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, U.K.
authors.forename: D J
authors.initials: DJ
authors.lastname: Meyer
authors.forename: C
authors.initials: C
authors.lastname: Xia
authors.forename: B
authors.initials: B
authors.lastname: Coles
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: P
authors.initials: P
authors.lastname: Reinemer
authors.forename: R
authors.initials: R
authors.lastname: Huber
authors.forename: B
authors.initials: B
authors.lastname: Ketterer
mesh_terms: D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D005260:Female; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000201:enzymology; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D006639:Histidine; D005982:Glutathione Transferase; D004047:Diethyl Pyrocarbonate