Photoaffinity labelling of methyltransferase enzymes with S-adenosylmethionine: effects of methyl acceptor substrates.
Radioactivity from 3H-[methyl]-S-adenosyl-L-methionine (AdoMet) was covalently bound to protein-O-carboxylmethyltransferase and phenylethanolamine N-methyltransferase following 10-15 min irradiation by short-wave ultraviolet light. This photoaffinity binding of 3H-[methyl]-AdoMet was blocked by S-adenosylhomocysteine and sinefungin, but was not affected by 5 mM dithiothreitol. The binding was also inhibited by including methyl acceptors such as calmodulin (protein-O-carboxylmethyltransferase) or phenylethanolamine (phenylethanolamine N-methyltransferase) in the photoaffinity incubation. Staphlococcus V8 protease digests of 3H-[methyl]-AdoMet/enzyme complexes revealed that the primary structure around the AdoMet binding site is different in these two enzymes. Thus, protein-O-carboxylmethyltransferase, a large molecule methyltransferase, can covalently bind 3H-[methyl]-AdoMet in a manner similar to that of phenylethanolamine-N-methyltransferase.
D000345:Affinity Labels; D001665:Binding Sites; D010450:Endopeptidases; D007700:Kinetics; D010625:Phenylethanolamine N-Methyltransferase / Q000378:metabolism* / Q000528:radiation effects; D011485:Protein Binding; D011496:Protein Methyltransferases / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism* / Q000528:radiation effects; D012697:Serine Endopeptidases; D014316:Tritium; D014466:Ultraviolet Rays
D000345:Affinity Labels; D014316:Tritium; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase; D010625:Phenylethanolamine N-Methyltransferase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase