Amino acid differences at positions 10, 11, and 104 explain the profound catalytic differences between two murine pi-class glutathione S-transferases.
The glutathione S-transferases play a pivotal role in the detoxification of toxic and carcinogenic electrophiles. We have previously reported the isolation of two actively transcribed murine pi-class glutathione S-transferase genes. In this study the two proteins encoded by these genes, Gst p-1 and Gst p-2, were expressed in Escherichia coli and found to exhibit profoundly different catalytic activities, the activity of Gst p-2 toward a panel of electrophilic substrates being 1-3 orders of magnitude lower than that of Gst p-1. In order to establish the basis for the difference between these highly homologous proteins, mutants were generated where specific amino acids had been exchanged. Kinetic analysis of the wild-type and mutant enzymes revealed that the amino acid differences occurring at positions 10 (Val/Ser), 11 (Arg/Pro), and 104 (Val/Gly) are responsible for the reduced enzymatic activity of Gst p-2. This analysis together with computer graphics modeling for Gst p-2 indicated that these changes affected both substrate and glutathione binding to the enzyme.
Bammler
T K
TK
Molecular Pharmacology Unit, Ninewells Hospital and Medical School, Dundee, Scotland.
Driessen
H
H
Finnstrom
N
N
Wolf
C R
CR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000235:genetics; D005982:Glutathione Transferase / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D005982:Glutathione Transferase