Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.
The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.
Wang
R W
RW
Department of Animal & Exploratory Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065-0900.
Newton
D J
DJ
Huskey
S E
SE
McKeever
B M
BM
Pickett
C B
CB
Lu
A Y
AY
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005453:Fluorescence; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D009841:Oligonucleotides; D013438:Sulfhydryl Compounds; D001224:Aspartic Acid; D014443:Tyrosine; D005982:Glutathione Transferase