Identification of the fatty acid binding site on glutathione S-transferase P by immobilization to fatty acid-linked sepharose.
The association of glutathione S-transferase P (GST-P) with various fatty acids was confirmed by gas-liquid chromatography and mass-spectrometry. Palmitic and stearic acids were the major fatty acids bound to the enzyme in which the molar ratio was 1:0.8 (GST-P:fatty acid). To evaluate the association with respect to the fatty acid carbon chain length and identify the binding site, we prepared a series of fatty acid-linked Sepharoses. GST-P tightly bound the fatty acid-linked Sepharoses (CH3(CH2)nCOOH, n = 4 approximately 16), and the site was determined to be residues 121-156 from the amino terminus by tryptic digestion of GST-P bound to a fatty acid-linked Sepharose. By fatty acid affinity labeling, we identified the binding site as residues 141-188 (Biochem. Biophys. Res. Commun., in press). The overlapping hydrophobic residues (residue 141-156) is expected to be essential for the ligand binding site.
Nishihira
J
J
Department of Biochemistry, School of Medicine, Hokkaido University, Sapporo, Japan.
Ishibashi
T
T
Sakai
M
M
Nishi
S
S
Kumazaki
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D005227:Fatty Acids; D010455:Peptides; D011994:Recombinant Proteins; D005982:Glutathione Transferase