Binding of C5-dicarboxylic substrate to aspartate aminotransferase: Implications for the conformational change at the transaldimination step
Binding of C5-dicarboxylic substrate to aspartate aminotransferase: Implications for the conformational change at the transaldimination step
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DOI：
10.1021/bi050071g
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发表时间：
2005-06-14
期刊：
影响因子：
2.9
通讯作者：
Hayashi, H
中科院分区：
文献类型：
--
作者：
Islam, MM;Goto, M;Hayashi, H
天冬氨酸氨基转移酶与C4底物L-天冬氨酸的反应机制已得到充分确立。C4底物的结合诱导酶从开放形式到封闭形式的构象变化，并且整个反应以酶的封闭形式进行。相反，人们对与C5底物L-谷氨酸的反应知之甚少。在这项研究中，我们分析了2-甲基-L-谷氨酸与酶的pH依赖性结合，并表明2-甲基-L-谷氨酸的氨基与吡哆醛5 '-磷酸醛亚胺之间的相互作用弱于2-甲基-L-天冬氨酸与醛亚胺之间的相互作用。酶与L-天冬氨酸和L-谷氨酸的米氏复合物的结构是在酶的马来酸和戊二酸复合物结构的基础上建模的。结果表明，L-谷氨酸以伸展构象与酶的开放形式结合，其α-氨基指向与醛亚胺相反的方向。而L-天冬氨酸与醛亚胺接近。这些模型解释了2-甲基-L-戊二酸和2-甲基-L-天冬氨酸的观察结果。本文以L-谷氨酸、D-谷氨酸和2-甲基-L-谷氨酸的磷酸吡哆衍生物与天冬氨酸氨基转移酶形成的复合物为模型，求解了它们的晶体结构。所有的结构都是封闭的，并且两个羧酸基团和与它们结合的精氨酸残基可以重叠在2-甲基-L-天冬氨酸的醛亚胺复合物上。综合这些事实，强烈地表明L-谷氨酸与天冬氨酸转氨酶结合形成米氏复合物不会引起酶的构象变化，并且在transaldimination步骤中发生了闭合形式的构象变化。活性位点入口处的疏水残基，包括Tyr 70，被认为对于促进transaldimination过程以及因此对C5底物的识别是重要的。
The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, L-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate L-glutamate. In this Study, we analyzed the pH-dependent binding of 2-methyl-L-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-L-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-L-aspartate and the aldimine. The Structures of the Michaelis complexes of the enzyme with L-aspartate and L-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that L-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine. while that of L-aspartate is close to the aldimine. These models explain the observations for 2-methyl-L-glutarnate and 2-methyl-L-aspartate. The crystal structures of the complexes of aspartate aiminotransferase with phosphopyridoxyl derivatives of L-glutamate, D-glutamate, and 2-methyl-L-glutamate were solved as the models for the external aldimine and ketimine complexes Of L-glutamate, All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-L-aspartate. Taking these facts altogether, it was strongly suggested that the binding Of L-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are considered to be important for promoting the transaldimination process and hence the recognition of the C5 substrate.