Identification of the hydrophobic ligand-binding region in recombinant glutathione S-transferase P and its binding effect on the conformational state of the enzyme.
Recombinant glutathione S-transferase P (GST-P) was purified in a homogeneous state. Fatty acid analysis of the enzyme revealed that the final enzyme preparation endogenously bound fatty acids, mostly palmitic acid or stearic acid, which were difficult to dissociate from the complex. Temperature-dependent analysis by 1H NMR indicated that the molecular motion of fatty acids was strongly restrained under physiological conditions, which was significantly different from that of serum albumin. On the other hand, there existed another hydrophobic ligand-binding region in GST-P, to which 1-amino-8-naphthalenesulfonic acid and bilirubin would bind with relatively lower affinity than the endogenously bound fatty acid. The hydrophobic ligand-binding region was determined to be around 141-156 residues from the N-terminus by procedures including association of the enzyme to fatty acid-linked Sepharose and affinity labeling with fluorescent fatty acid. Furthermore, circular dichroism analysis showed that the binding of hydrophobic ligand to GST-P produced a remarkable conformational change of the enzyme, which led to states devoid of transferase activity. In addition, the hydrophobic ligand binding caused a significant fluorescence quenching of tryptophan 38, which was assumed to be located at the active center of GST-P. It could be the result of a conformational change of the active center of the enzyme.
Nishihira
J
J
Department of Biochemistry, School of Medicine, Hokkaido University, Sapporo, Japan.
Ishibashi
T
T
Sakai
M
M
Tsuda
S
S
Hikichi
K
K
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001663:Bilirubin / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013229:Stearic Acids / Q000378:metabolism
D000345:Affinity Labels; D005227:Fatty Acids; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010169:Palmitic Acids; D011994:Recombinant Proteins; D013229:Stearic Acids; C031762:1-naphthylamine-8-sulfonic acid; D019308:Palmitic Acid; C031183:stearic acid; D005982:Glutathione Transferase; D001663:Bilirubin