title: A highly acidic tyrosine 9 and a normally titrating tyrosine 212 contribute to the catalytic mechanism of human glutathione transferase A4-4.
abstract: Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and other alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy and site-directed mutagenesis. The titration curve of GST A4-4 shows a pK(a) of 6.7 attributable to tyrosine 9, which in the Y212F mutant was shifted to pK(a) 7.1. In both cases the pK(a) was independent of the absence or presence of GSH. Thus, the active-site tyrosine 9 of this isoenzyme is more than one unit more acidic than the corresponding tyrosine of other Alpha class glutathione transferases. The tyrosines remaining in the Y9F mutant titrate like free tyrosine with pK(a) values > or = 10. A mechanism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4.
authors.affiliation: Department of Biochemistry, Uppsala University, Biomedical Center, SE-751 23 Uppsala, Sweden.
authors.forename: I
authors.initials: I
authors.lastname: Hubatsch
authors.forename: B
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D014867:Water; D014443:Tyrosine; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase