Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine.
A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide.
D000123:Acetyltransferases / Q000032:analysis*; D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D001386:Azides; D002331:Carnitine / Q000031:analogs & derivatives*; D002332:Carnitine O-Acetyltransferase / Q000032:analysis* / Q000037:antagonists & inhibitors; D008623:Mercaptoethanol / Q000494:pharmacology; D010449:Peptide Mapping; D010777:Photochemistry; D013053:Spectrophotometry
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; C050881:4-azidophenacetylthiocarnitine; D008623:Mercaptoethanol; D000123:Acetyltransferases; D002332:Carnitine O-Acetyltransferase; D002331:Carnitine