Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione.
In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.
Manoharan
T H
TH
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Gulick
A M
AM
Puchalski
R B
RB
Servais
A L
AL
Fahl
W E
WE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics
D000596:Amino Acids; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D005982:Glutathione Transferase; D001618:beta-Lactamases; D005978:Glutathione