Covalently modified peptides isolated from aspartate aminotransferase after reaction with pyridoxal 5'-sulfate.
The apoenzyme form of cytosolic aspartate aminotransferase of pig hearts was allowed to react at room temperature with 1 equiv of pyridoxal 5'-sulfate. The resulting covalently modified enzyme was degraded with pepsin. Fluorescent tri-, tetra-, and hexapeptides were isolated and characterized as fragments of the active site sequence: Phe-Ser-Lys-Asn-Phe-Gly-Leu. This sequence contains a modified form (Lys) of lysine-258 that is known to form a Schiff base with pyridoxal phosphate in the active site. The peptides were further degraded by acid hydrolysis to give a fluorescent derivative of lysine with light absorption and chemical properties similar to those of the original modified enzyme. A related series of peptides were obtained from apoenzyme after reaction with the 5-carboxyethenyl analogue of pyridoxal 5'-phosphate.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001219:Aspartate Aminotransferases / Q000032:analysis*; D002268:Carboxypeptidases / Q000378:metabolism; D002850:Chromatography, Gel; D010446:Peptide Fragments / Q000302:isolation & purification*; D011402:Pronase / Q000378:metabolism; D011730:Pyridoxal / Q000031:analogs & derivatives* / Q000494:pharmacology; D013552:Swine
D000596:Amino Acids; D010446:Peptide Fragments; D011730:Pyridoxal; C036319:pyridoxal 5'-sulfate; D001219:Aspartate Aminotransferases; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D011402:Pronase