Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure

• Published in: Journal of molecular biology Vol. 174; no. 3; pp. 497 - 525
• Main Authors: Kirsch, Jack F., Eichele, Gregor, Ford, Geoffrey C., Vincent, Michael G., Jansonius, Johan N., Gehring, Heinz, Christen, Philipp
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 15.04.1984; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Aspartate Aminotransferases - metabolism; Aspartic Acid; Binding Sites; Biological and medical sciences; Coenzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glutamates; Glutamic Acid; Kinetics; Ligands; Models, Molecular; Protein Conformation; Substrate Specificity; Transferases; X-Ray Diffraction; Reaction intermediate; Pyridoxal; Molecular structure; Enzyme; Enzymatic catalysis; Binding site; Cofactor; Pyridoxal-phosphate enzyme; Coenzyme; Substrate; Vertebrata; Mitochondria; Electron density map; Reaction mechanism; Aspartate aminotransferase; Models; Chicken; Aves
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(84)90333-4

Aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme that catalyses the transamination reaction: l-aspartate + 2-oxoglutarate ⇋ oxaloacetate + l-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. This paper proposes a mechanism of action that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 Å resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogues, as well as on model building studies. The enzyme is composed of two identical subunits, which consist of two domains. The coenzyme is bound to the larger domain and is situated in a pocket near the subunit interface. The proximal and distal carboxylate group of dicarboxylic substrates are bound to Arg386 and Arg292, respectively, the latter residue belonging to the adjacent subunit. These interactions largely determine the substrate specificity of the enzyme. They not only position the substrate for efficient catalysis but also bring about a bulk movement of the small domain that closes the active site crevice and moves Arg386 about 3 Å closer to the coenzyme. The replacement of the ε-amino group of Lys258 by the α-amino group of the substrate in the aldimine bond to pyridoxal phosphate is accompanied by a tilting of the coenzyme by ~30 °. The released ε-amino group of Lys258 serves as a proton acceptor/donor in the 1,3-prototropic shift producing the ketimine intermediate. At this stage, or after hydrolysis of the ketimine bond, the coenzyme rotates back to an orientation between that in the “external” aldimine intermediate and that in the pyridoxal form. Throughout this process, the protonated pyridine nitrogen atom maintains a hydrogen bond to the β-carboxylate group of Asp222. Upon formation of the pyridoxamine form, the small domain moves back to its original position. The proposed mechanism is compatible with the known kinetic and stereochemical features of enzymic transamination.