Aspartate aminotransferase complexed with erythro-beta-hydroxyaspartate: crystallographic and spectroscopic identification of the carbinolamine intermediate.
The crystal structure of mitochondrial aspartate aminotransferase (mAAT) of chicken complexed with erythro-beta-hydroxyaspartate has been determined at 2.4 A resolution. Pregrown crystals of mAAT complexed with the inhibitor maleate (closed enzyme conformation, orthorhombic space group C222(1)) were soaked in solutions of erythro-beta-hydroxyaspartate. The ligand exchange was monitored by microspectrophotometry. The active site turned out to be predominantly occupied by the carbinolamine intermediate. The carbinolamine is a true intermediate of the catalytic cycle forming the last covalently bound enzyme:substrate complex before release of the keto acid product. Occupancies of approximately 80% for the carbinolamine and of approximately 20% for the quinonoid intermediate were obtained. Two hydrogen bonds were identified that are potentially relevant for the accumulation of the carbinolamine intermediate: one to the hydroxyl group of Tyr 70* and the other to the epsilon-NH2 group of Lys 258.
von Stosch
A G
AG
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011487:Protein Conformation; D013237:Stereoisomerism
D000900:Anti-Bacterial Agents; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D001219:Aspartate Aminotransferases