Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes.
Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes.
复制标题
大肠杆菌天冬氨酸转氨酶的定点诱变：Tyr70 在催化过程中的作用。
作者：
K. Inoue;S. Kuramitsu;A. Okamoto;K. Hirotsu;T. Higuchi;H. Kagamiyama
对大肠杆菌天冬氨酸氨基转移酶（AspAT）活性位点Tyr 70进行定点突变，并进行动力学研究，阐明了Tyr 70的羟基和苯环的作用。X-射线晶体学分析表明，取代Tyr 70苯丙氨酸没有改变酶的活性位点构象。四种半转氨反应动力学参数的比较（酶的吡哆醛5 ′-磷酸形式与L-天冬氨酸或L-谷氨酸和吡哆胺5 ′-磷酸形式与N-乙酰乙酸或2-酮戊二酸）之间的比较表明，突变使所有四种底物的过渡态能级增加了2 kcal.mol-1，表明Tyr 70的羟基对过渡态有一定的贡献。当Phe 70进一步被Ser取代时，L-谷氨酸或2-酮戊二酸的过渡态能级进一步增加了2-3 kcal.mol-1，这表明在位置70处存在苯环对于识别L-谷氨酸-2-酮戊二酸对作为底物是必不可少的。
Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70. X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme. Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and [Tyr70----Phe]AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state. When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.
酪氨酸 70 增加天冬氨酸转氨酶的辅酶亲和力。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Toney,MD;Kirsch,JF
通讯作者：
Kirsch,JF