PARTICIPATION OF THE PHENOLIC HYDROXYL GROUP OF TYR-8 IN THE CATALYTIC MECHANISM OF HUMAN GLUTATHIONE TRANSFERASE P1-1

The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized. The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta. The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis. The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value. Kinetic parameters reflecting binding affinity, S0.5 (substrate concn. giving 1/2 V) and I50 (concn. of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme. These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates. The dependence of k(cat.)/K(m) on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pK(a1) = 6.7 and pK(a2) = 7.3 respectively. The mutant enzyme Y8F does not display the basic limb of the k(cat.)/K(m) versus pH profile, but shows a monotonic increase of k(cat.)/K(m) with an apparent pK(a1) of 7.1. The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1.