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 1/2V) 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.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively. The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 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.
Kolm
R H
RH
Department of Biochemistry, University of Uppsala, Sweden.
Sroga
G E
GE
Mannervik
B
B
D001483:Base Sequence; D002384:Catalysis; D004247:DNA; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics*
D011994:Recombinant Proteins; D014443:Tyrosine; D004247:DNA; D005982:Glutathione Transferase