Human glutathione transferase A1-1 demonstrates both half-of-the-sites and all-of-the-sites reactivity.
A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.
Lien
S
S
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
Gustafsson
A
A
Andersson
A K
AK
Mannervik
B
B
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha