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Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3.

ABSTRACT: Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.

SUBMITTER: Chen WL

PROVIDER: S-EPMC1133040 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Project description:Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis. Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala. The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised. Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties. All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB). Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione. In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST. With the substrates used, kcat./Km values were decreased for all mutant GSTs. Increases in the [S0.5] values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected. Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities. Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A. Mutant R20A had a lowered [I50] value but, in contrast, also the highest [I80] value as compared with the wild-type enzyme. Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type. The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between. The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations. The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST. Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.

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Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3.

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