[Identification and characterization of hydrophobic ligand binding region in glutathione S-transferase P].
Recombinant glutathione S-transferase P (GST-P) was purified in a homogeneous state. Fatty acid analysis by gas-liquid chromatography-mass spectrometry (GC-MS) revealed that GST-P forms 1:1 complex with fatty acids, mostly palmitic acid or stearic acid, which were hardly isolated from the complex even through Lipidex 1,000 column chromatography at 37 degrees C. Temperature dependent analysis of 1H-NMR on the association between GST-P and fatty acids indicated that molecular motion of fatty acids were strongly restrained in a hydrophobic 'pocket' below the temperature of protein denaturation. On the other hand, there existed another hydrophobic ligand binding region, to which fatty acid and bilirubin would bind with relatively lower affinity. The binding region was determined to be at around 142-157 residues from amino terminus by the studies of GST-P binding to fatty acid-linked Sepharose and affinity labelings with either fluorescent fatty acid or bilirubin. The binding to this region noncompetitively inhibited the enzyme activity. Furthermore, circular dichroism (CD) analysis showed that the binding of hydrophobic ligands changed the secondary structure of GST-P, which suggested that the enzyme activity was regulated through conformational changes. As tryptophan 38 was assumed to locate at the active center from the study of site-directed mutagenesis, conformation of the active center was investigated by measuring the intrinsic tryptophan fluorescence. It showed that hydrophobic ligand binding caused the drastic conformational change, of which would be referred to the regulation of the enzyme activity.
Nishihira
J
J
Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Japan.
D001665:Binding Sites; D002942:Circular Dichroism; D005227:Fatty Acids / Q000032:analysis; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D008024:Ligands; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000032:analysis
D005227:Fatty Acids; D008024:Ligands; D011994:Recombinant Proteins; D005982:Glutathione Transferase