Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis.
To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.
Tamai
K
K
Second Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Shen
H X
HX
Tsuchida
S
S
Hatayama
I
I
Satoh
K
K
Yasui
A
A
Oikawa
A
A
Sato
K
K
D000818:Animals; D001483:Base Sequence; D003538:Cystamine / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D003545:Cysteine; D005033:Ethylmaleimide; D003538:Cystamine