Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi.
Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.
Manoharan
T H
TH
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Gulick
A M
AM
Reinemer
P
P
Dirr
H W
HW
Huber
R
R
Fahl
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003461:Crystallography; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000648:ultrastructure*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013552:Swine
C040136:glutathione sulfonate; D005982:Glutathione Transferase; D005978:Glutathione