Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3.
Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.
Johansson
Ann-Sofie
AS
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, Uppsala SE-75123, Sweden.
Mannervik
Bengt
B
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D013251:Steroid Isomerases / Q000737:chemistry; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D013438:Sulfhydryl Compounds; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D005978:Glutathione