Identification of the fatty acid binding site on glutathione S-transferase P.
Glutathione S-transferase P (GST-P) bound a series of endogenous fatty acids (C12-C18). To clarify the function and the binding site of the fatty acids, interaction between fatty acids and GST-P was investigated by using 12-(9-anthroyloxy) stearic acid conjugated with Woodward's reagent K. The fluorescence-conjugated fatty acid noncompetitively inhibited GST activity. After GST-P was covalently labeled with the fatty acid, the enzyme was digested with Lysyl Endopeptidase. From the peptide mapping, a single fluorescence-labeled peptide was obtained. By the sequence analysis, the peptide binding fatty acid was determined as the residues of 141-188 from the amino terminus.
Nishihira
J
J
Department of Biochemistry, Hokkaido University, Sapporo, Japan.
Ishibashi
T
T
Sakai
M
M
Nishi
S
S
Kondo
H
H
Makita
A
A
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism*; D005456:Fluorescent Dyes; D008401:Gas Chromatography-Mass Spectrometry; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013229:Stearic Acids / Q000378:metabolism*
D000345:Affinity Labels; D005227:Fatty Acids; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011994:Recombinant Proteins; D013229:Stearic Acids; C030310:12-(9-anthroyl)stearic acid; D005982:Glutathione Transferase