Kirsch J F, Eichele G, Ford G C, Vincent M G, Jansonius J N, Gehring H, Christen P
J Mol Biol. 1984 Apr 15;174(3):497-525. 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 A 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 efficient catalysis but also bring about a bulk movement of the small domain that closes the active site crevice and moves Arg386 about 3 A closer to the coenzyme. The replacement of the epsilon-amino group of Lys258 by the alpha-amino group of the substrate in the aldimine bond to pyridoxal phosphate is accompanied by a tilting of the coenzyme by approximately 30 degrees. The released epsilon-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 beta-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.
天冬氨酸转氨酶是一种依赖磷酸吡哆醛的酶，它催化转氨反应：L-天冬氨酸 + 2-氧代戊二酸→草酰乙酸 + L-谷氨酸。该酶在双置换过程中在其磷酸吡哆醛和磷酸吡哆胺形式之间穿梭。本文提出了一种作用机制，阐述了该酶蛋白质部分的动态作用。它基于线粒体同工酶在无配体形式以及与底物类似物形成的复合物中的晶体学确定的空间结构（分辨率为2.8埃），以及模型构建研究。该酶由两个相同的亚基组成，每个亚基由两个结构域组成。辅酶与较大的结构域结合，位于亚基界面附近的一个口袋中。二羧酸底物的近端和远端羧基分别与Arg386和Arg292结合，后者属于相邻亚基。这些相互作用在很大程度上决定了酶的底物特异性。它们不仅使底物定位以进行有效催化，还引起小结构域的大量移动，从而关闭活性位点裂缝并使Arg386向辅酶靠近约3埃。在与磷酸吡哆醛的醛亚胺键中，底物的α-氨基取代Lys258的ε-氨基时，辅酶会倾斜约30度。释放的Lys258的ε-氨基在产生酮亚胺中间体的1,3-质子转移中作为质子受体/供体。在这个阶段，或者在酮亚胺键水解之后，辅酶旋转回到“外部”醛亚胺中间体和磷酸吡哆醛形式之间的取向。在整个过程中，质子化的吡啶氮原子与Asp222的β-羧基保持氢键。形成磷酸吡哆胺形式后，小结构域回到其原始位置。所提出的机制与酶促转氨作用已知的动力学和立体化学特征相符。
