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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Translational misreading, amino acid misincorporation and misinterpretations. The case of the flavocytochrome b(2) H373Q variant
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Translational misreading, amino acid misincorporation and misinterpretations. The case of the flavocytochrome b(2) H373Q variant

机译：平移误读，氨基酸的MISlincororation和误解。 黄鳞菌菌B（2）H373Q变体的情况

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摘要

Amino acid misincorporation during protein synthesis occurs naturally at a low level. Protein sequence errors, depending on the level and the nature of the misincorporation, can have various consequences. When site -directed mutagenesis is used as a tool for understanding the role of a side chain in enzyme catalysis, misincorporation in a variant with intrinsically low activity may lead to misinterpretations concerning the enzyme mechanism. We report here one more example of such a problem, dealing with flavocytochrome b(2) (Fcb2), a lactate dehydrogenase, member of a family of FMN-dependent L-2-hydroxy acid oxidizing enzymes. Two papers have described the properties of the Fcb2 catalytic base H373Qvariant, each one using a different expression system with the same base change for the mutation. The two papers found similar apparent kinetic parameters. But the first one demonstrated the existence of a low level of histidine misincorporation, which led to an important correction of the variant residual activity (Gaume et al. (1995) Biochimie, 77, 621). The second paper did not investigate the possibility of a misincorporation (Tsai et al. (2007) Biochemistry, 46, 7844). The two papers had different mechanistic conclusions. We show here that in this case the misincorporation does not depend on the expression system. We bring the proof that Tsai et al. (2007) were led to an erroneous mechanistic conclusion for having missed the phenomenon as well as for having misinterpreted the crystal structure of the variant. This work is another illustration of the caution one should exercise when characterizing enzyme variants with low activity. (C) 2016 Elsevier B.V. All rights reserved.
机译：蛋白质合成期间的氨基酸MIS掺入天然存在于低水平。蛋白质序列误差，取决于MISINCORANTION的水平和性质，可以具有各种后果。当网站 - 分界的诱变用作理解酶催化中侧链的作用时，具有本质上低活性的变体中的MIRINCORATION可能导致关于酶机制的误解。我们在这里报告这种问题的另一个例子，处理黄酮酵母B（2）（Fcb2），乳酸脱氢酶，依赖于FMN依赖性L-2-羟基酸氧化酶的成员。两篇论文已经描述了FCB2催化基质H373 QVariant的性质，每个纸张使用不同的表达系统，每个表达系统具有相同的突变变化。两篇论文发现了类似的表观动力学参数。但第一个证明存在低水平的组氨酸的MISINCORNATION，这导致了变异残留活性的重要校正（GAUME等，（1995）Biochimie，77,621）。第二篇论文没有调查MISINCORNATION（TSAI等）的可能性（Tsai等人，2007年）生物化学，46,7844）。这两篇论文的机制结论不同。我们在此显示在这种情况下，MISINCORNATION不依赖于表达式系统。我们带来了Tsai等人的证据。 （2007年）导致了一个错误的机制结论，以错过了这种现象以及误解了变体的晶体结构。这项工作是在表征具有低活性的酶变体时应锻炼的警告的另一插图。 （c）2016年Elsevier B.v.保留所有权利。

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