Lue Stanley W, Kelley Shana O
Eugene F. Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Biochemistry. 2005 Mar 1;44(8):3010-6. doi: 10.1021/bi047901v.
Many aminoacyl-tRNA synthetases (aaRSs) contain two active sites, a synthetic site catalyzing aminoacyl-adenylate formation and tRNA aminoacylation and a second editing or proofreading site that hydrolyzes misactivated adenylates or mischarged tRNAs. The combined activities of these two sites lead to rigorous accuracy in tRNA aminoacylation, and both activities are essential to LeuRS and other aaRSs. Here, we describe studies of the human mitochondrial (hs mt) LeuRS indicating that the two active sites of this enzyme have undergone functional changes that impact how accurate aminoacylation is achieved. The sequence of the hs mt LeuRS closely resembles a bacterial LeuRS overall but displays significant variability in regions of the editing site. Studies comparing Escherichia coli and hs mt LeuRS reveal that the proofreading activity of the mt enzyme is disrupted by these sequence changes, as significant levels of Ile-tRNA(Leu) are formed in the presence of high concentrations of the noncognate amino acid. Experiments monitoring deacylation of Ile-tRNA(Leu) and misactivated adenylate turnover revealed that the editing active site is not operational. However, hs mt LeuRS has weaker binding affinities for both cognate and noncognate amino acids relative to the E. coli enzyme and an elevated discrimination ratio. Therefore, the enzyme achieves fidelity using a more specific synthetic active site that is not prone to errors under physiological conditions. This enhanced specificity must compensate for the presence of a defunct editing site and ensures translational accuracy.
许多氨酰 - tRNA合成酶（aaRSs）含有两个活性位点，一个是催化氨酰 - 腺苷酸形成和tRNA氨酰化的合成位点，另一个是编辑或校对位点，可水解错误激活的腺苷酸或错误负载的tRNA。这两个位点的联合活性导致tRNA氨酰化具有高度准确性，且这两种活性对亮氨酰 - tRNA合成酶（LeuRS）和其他aaRSs都至关重要。在此，我们描述了对人线粒体（hs mt）LeuRS的研究，结果表明该酶的两个活性位点发生了功能变化，影响了实现准确氨酰化的方式。hs mt LeuRS的序列总体上与细菌LeuRS非常相似，但在编辑位点区域表现出显著差异。比较大肠杆菌和hs mt LeuRS的研究表明，这些序列变化破坏了线粒体酶的校对活性，因为在高浓度非同源氨基酸存在的情况下会形成大量的异亮氨酰 - tRNA（Leu）。监测异亮氨酰 - tRNA（Leu）的去酰化和错误激活的腺苷酸周转的实验表明，编辑活性位点不起作用。然而，相对于大肠杆菌酶，hs mt LeuRS对同源和非同源氨基酸的结合亲和力较弱，且歧视率升高。因此，该酶通过一个在生理条件下不易出错的更具特异性的合成活性位点来实现保真度。这种增强的特异性必须弥补失活编辑位点的存在，并确保翻译准确性。
