Identification of cysteine residues involved in disulfide formation in the inactivation of glutathione transferase P-form by hydrogen peroxide.
We previously reported that rat glutathione transferase P-form (GST-P) is inactivated by hydrogen peroxide (H2O2). This involves formation of intra- or intersubunit disulfides, at least three extra bands with molecular masses of 21.5, 18, and 37 kDa being exhibited in addition to the native subunit band of 23.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In the present study, GST-P mutants whose cysteine residues were independently substituted with alanine (C14A, C47A, C101A, and C169A) by site-directed mutagenesis were used to identify the cysteine residues responsible for the disulfide bond formation. C14A and C169A were much more inactivated than native GST-P by 1 mM H2O2, whereas C47A and, especially, C101A appeared insensitive to H2O2. On SDS-PAGE, the 21.5-kDa band was not detected in either C47A or C101A. Hydrogen peroxide treatment of mouse GST II, highly homologous to rat GST-P but possessing glycine instead of cysteine at the 101st residue, did not result in generation of the 21.5-kDa band and was also associated with less inactivation. This band was therefore considered to be due to an intrasubunit disulfide bond between Cys-47 and Cys-101. The 37-kDa band was suggested to be due to the formation of intersubunit disulfide bonds between Cys-47 residues in different subunits. Thus the Cys-47 residue together with Cys-101 may be located in an important region for GSH binding, disulfide bond formation between these residues resulting in steric hindrance.
Shen
H
H
Second Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Tsuchida
S
S
Tamai
K
K
Sato
K
K
D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms / Q000201:enzymology; D008970:Molecular Weight; D011230:Precancerous Conditions / Q000201:enzymology; D051381:Rats
D004220:Disulfides; D007527:Isoenzymes; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine; C033550:hexylglutathione