Reversible modification of rat liver glutathione S-transferase 3-3 with 1-chloro-2,4-dinitrobenzene: specific labelling of Tyr-115.
Rat liver glutathione S-transferase 3-3 (GST, EC 2.5.1.18), a triple mutant with all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were overexpressed in Escherichia coli under the control of a phoA promoter. Using this system, we obtained over 35 mg of fully active pure protein/litre of cell medium. GST 3-3 and CallS mutant were modified with 1-chloro-2,4-dinitrobenzene (CDNB), a model substrate for the enzyme, in the absence of GSH. Dinitrophenol, but not S-methylglutathione, inhibits this process. The dinitrophenyl groups are readily removed from the enzyme with GSH, but much more slowly with dithiothreitol. Results from peptide mapping and amino acid sequence analyses indicate that CDNB modifies the cysteine residues and Tyr-115 on wild-type GST 3-3, but only Tyr-115 on CallS. In addition, CDNB cannot modify the CallSY115F mutant. We propose that Tyr-115 is located at or near the H-site of GST 3-3.
Liu
L F
LF
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Hong
J L
JL
Tsai
S P
SP
Hsieh
J C
JC
Tam
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004137:Dinitrochlorobenzene / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014443:Tyrosine
D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; D005982:Glutathione Transferase