Site-directed mutagenesis of glutathione S-transferase YaYa: nonessential role of histidine in catalysis.
A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.
Wang
R W
RW
Department of Animal & Exploratory Drug Metabolism, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065.
Newton
D J
DJ
Pickett
C B
CB
Lu
A Y
AY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006639:Histidine; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D004247:DNA; D005982:Glutathione Transferase