Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation.
Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation.
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来自面包酵母的异亮酰-tRNA 合成酶：以调节精度区分异亮氨酸和缬氨酸的多步校对，这是一种通过能量耗散进行分子识别的方案。
DOI：
10.1021/bi00345a040
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
F. Cramer
中科院分区：
文献类型：
--
作者：
W. Freist;I. Pardowitz;F. Cramer
为了用酵母异亮氨酰-tRNA合成酶区分异亮氨酸和缬氨酸，建立了一个多步序列。最初的歧视的基板是继预转移和后转移水解校对过程。总区分因子D由在tRNAIle-C-C-A与异亮氨酸和缬氨酸的氨酰化中观察到的kcat和Km值确定。从修饰的tRNA种类tRNAIle-C-C-3 'dA和tRNAIle-C-C-A（3' NH 2）的氨酰化，可以确定初始辨别因子I（对可逆底物结合有效）和校正因子P1（对氨酰腺苷酸形成有效）。因子I从ATP消耗和D1计算，D1是可以从动力学常数获得的该部分反应的总体区分因子，P1从AMP形成速率计算。校正因子P2（对氨酰转移反应有效）由在tRNAIle-C-C-A和tRNAIle-C-C-3 ′ dA的氨酰化中观察到的AMP形成速率确定。由初始辨别因子I和AMP形成速率，可以计算tRNAIle-C-C-A的氨酰化中的辨别因子DAMP。这些值与通过动力学获得的因子D相差因子II，这可能是由于对于tRNAIle-C-C-A的酰化，初始区分因子I' = III是有效的。观察到的总体歧视根据条件变化高达16倍。在最佳条件下，每一个错误产生38000个正确的氨酰-tRNA，而消耗5.5个ATP的能量。随着确定的能量和分子流的酶反应的各个步骤，这种新型的“远离平衡酶”的连贯画面出现。
For discrimination between isoleucine and valine by isoleucyl-tRNA synthetase from yeast, a multistep sequence is established. The initial discrimination of the substrates is followed by a pretransfer and a posttransfer hydrolytic proofreading process. The overall discrimination factor D was determined from kcat and Km values observed in aminoacylation of tRNAIle-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNAIle-C-C-3'dA and tRNAIle-C-C-A (3'NH2), the initial discrimination factor I (valid for the reversible substrate binding) and the proofreading factor P1 (valid for the aminoacyl adenylate formation) could be determined. Factor I was computed from ATP consumption and D1, the overall discrimination factor for this partial reaction which can be obtained from kinetic constants, and P1 was calculated from AMP formation rates. Proofreading factor P2 (valid for aminoacyl transfer reaction) was determined from AMP formation rates observed in aminoacylation of tRNAIle-C-C-A and tRNAIle-C-C-3'dA. From the initial discrimination factor I and the AMP formation rates, discrimination factor DAMP in aminoacylation of tRNAIle-C-C-A can be calculated. These values deviate by a factor II from factor D obtained by kinetics which may be due to the fact that for acylation of tRNAIle-C-C-A an initial discrimination factor I' = III is valid. The observed overall discrimination varies up to a factor of 16 according to conditions. Under optimal conditions, 38 000 correct aminoacyl-tRNAs are produced per 1 error while the energy of 5.5 ATPs is dissipated. With the determined energetic and molecular flows for the various steps of the enzymatic reaction, a coherent picture of this new type of "far away from equilibrium enzyme" emerges.