MECHANISM OF ACTION OF ASPARTATE-AMINOTRANSFERASE PROPOSED ON THE BASIS OF ITS SPATIAL STRUCTURE
MECHANISM OF ACTION OF ASPARTATE-AMINOTRANSFERASE PROPOSED ON THE BASIS OF ITS SPATIAL STRUCTURE
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DOI：
10.1016/0022-2836(84)90333-4
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发表时间：
1984-01-01
影响因子：
5.6
通讯作者：
CHRISTEN, P
中科院分区：
文献类型：
--
作者：
KIRSCH, JF;EICHELE, G;CHRISTEN, P
天冬氨酸氨基转移酶[鸡心]是一种磷酸吡哆醛依赖性酶，催化转氨反应：L-天冬氨酸+ 2-酮戊二酸。草酰乙酸+L-谷氨酸。该酶在其吡哆醛和吡哆胺形式之间以双置换过程穿梭。提出了一种作用机制，描绘了这种酶的蛋白质部分的动态作用。它是基于晶体学确定的空间结构（在2.8埃。分辨率）的线粒体同工酶在其unliganded形式和与底物类似物的复合物，以及模型构建研究。该酶由2个相同的亚基组成，亚基由2个结构域组成。辅酶与较大的结构域结合，并位于亚基界面附近的口袋中。二羧酸底物的近端和远端羧酸基团分别与Arg 386和Arg 292结合，后者残基属于相邻亚基。这些相互作用在很大程度上决定了酶的底物特异性。它们不仅定位底物以进行有效催化，而且还引起小结构域的整体移动，其闭合活性位点缝隙并使Arg 386移动约3埃。更接近辅酶。ε的替换Lys 258的氨基通过α-醛亚胺键合到磷酸吡哆醛上的底物的氨基伴随着辅酶的倾斜Δ Px。30度释放的ε- Lys 258的氨基在产生酮亚胺中间体的1，3-质子转移中充当质子受体/供体。在这个阶段，或在酮亚胺键水解后，辅酶旋转回到外部醛亚胺中间体和吡哆醛形式之间的取向。在整个该过程中，质子化的吡啶N原子保持与β-N原子的H-键。Asp 222的羧基。在吡哆胺形式形成后，小结构域移回其原始位置。提出的机制与已知的酶促转氨作用的动力学和立体化学特征是相容的。
Aspartate aminotransferase [chicken heart] is a pyridoxal phosphate-dependent enzyme that catalyzes the transamination reaction: L-aspartate + 2-oxoglutarate .dblarw. oxaloacetate + L-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. A mechanism of action is proposed that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 .ANG. resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogs, as well as on model building studies. The enzyme is composed of 2 identical subunits, which consist of 2 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 .ANG. 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 .apprx. 30.degree.. The released .epsilon.-amino group of Lys258 serves 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 N atom maintains a H-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.