Determination by photoaffinity labelling of the hydrophobic part of the binding site for acyl-CoA esters on acyl-CoA-binding protein from bovine liver.
Acyl-CoA esters containing the photoreactive acids 12-(4'-azido-2'-nitrophenoxy)[1-14C]dodecanoic acid ([14C]AND-acid) or N-(4'-azido-2'-nitro-[3'-5'-3H]phenyl)-12-aminododecanoic acid ([3H]NANPA-acid) were synthesized. The photoreactive acyl-CoA esters could be bound to bovine acyl-CoA-binding protein (ACBP) and photocrosslinked to the protein. The photocrosslinked acyl-CoA-ACBP complex was separated from unlabelled ACBP on reverse-phase h.p.l.c. and the purified complex was digested with trypsin, Staphylococcus aureus V8 proteinase or endoproteinase Asp-N. By four independent peptide maps it was shown that the amino acids taking part in forming the hydrophobic binding site for acyl-CoA esters in bovine ACBP are located on the peptide segment from Asp21 to Asp38. Both photoreactive acyl-CoA esters used in this study labelled strongly in the segment from Tyr28 to Ala34. 12-(4'-Azido-2'-nitrophenoxy)[1-14C]-dodecanoyl-CoA ([14C]AND-CoA) also introduced a label at position Asp38, but o labelling was found before Ser29. In contrast, N-(4'-azido-2'-nitro[3',5'-3H]phenyl)-12-aminododecanoyl-CoA [3H]NANPA-CoA) also labelled the segment from Asp21 to Tyr28. The difference in labelling by the two photoreactive ligands is most likely caused by different mobility of the arylazido group when linked to the fatty acid either through a phenolic O- or an anilinic N- bond.
Hach
M
M
Institute of Biochemistry, Odense University, Denmark.
Pedersen
S N
SN
Börchers
T
T
Højrup
P
P
Knudsen
J
J
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D026261:Diazepam Binding Inhibitor; D010450:Endopeptidases; D007848:Laurates / Q000138:chemical synthesis / Q000378:metabolism; D008099:Liver / Q000737:chemistry*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D010777:Photochemistry; D012697:Serine Endopeptidases; D014357:Trypsin
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D026261:Diazepam Binding Inhibitor; D007848:Laurates; C065987:12-(4'-azido-2'-nitrophenoxy)dodecanoyl-coenzyme A; C065988:N-(4'-azido-2'-nitrophenyl)-12-aminododecanoyl-coenzyme A; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N