A highly acidic tyrosine 9 and a normally titrating tyrosine 212 contribute to the catalytic mechanism of human glutathione transferase A4-4

Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and oth er alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy an d 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 prese nce 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 greater than or equal to 10. A mechan ism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4. (C) 2001 Acade mic Press.