The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli. Evidence for a two-step aminoacylation pathway, and study of the reactivity of the intermediate complex.

D. Kern, J. Lapointe
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引用次数: 23

Abstract

The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAGlu aminoacylation by glutamyl-tRNA synthetase from Escherichia coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies. The nature of the kinetic patterns indicates that ATP and tRNAGlu bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme · tRNAGlu· ATP complex. Binding of ATP to the enzyme hinders that of tRNAGlu and vice versa. After interconversion of the quaternary enzyme · substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last. In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNAGlu promotes the catalytically active enzyme state. Whereas at saturating tRNAGlu concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAGlu concentrations. The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.
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大肠杆菌谷氨酰胺- trna合成酶的催化机制。两步氨基酰化途径的证据,以及中间复合物反应性的研究。
利用双底物动力学、终端和终端产物抑制研究,研究了大肠杆菌谷氨酰胺- trna合成酶催化tRNAGlu氨基酰化过程中底物结合和终端产物解离的顺序。动力学模式的性质表明,ATP和tRNAGlu随机结合到游离酶上,而谷氨酸只结合到三元酶·tRNAGlu·ATP复合物上。ATP与酶的结合阻碍了tRNAGlu的结合,反之亦然。在季酶·底物复合物相互转化后,最终产物按以下顺序解离:PPi首先,AMP其次,Glu-tRNA最后。除了作为底物和与ATP结合的效应物外,tRNAGlu还促进了催化活性酶的状态。而在饱和tRNAGlu浓度下,催化作用是速率决定的,在低tRNAGlu浓度下,这种构象变化可以是速率决定的。根据该合成酶催化的两步氨基酰化途径对结果进行了讨论。
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