Vu Michael T, Martinis Susan A
Department of Biochemistry, Roger Adams Laboratory, University of Illinois at Urbana-Champaign, Box B4, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Biochemistry. 2007 May 1;46(17):5170-6. doi: 10.1021/bi062078j. Epub 2007 Apr 4.
Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucine-specific domain is critical for aminoacylation but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts kcat. Because of its proximity to the aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the aminoacylation to the editing complex.
亮氨酰 - tRNA合成酶（LeuRS）是一种I类酶，其氨酰化活性位点位于由Rossmann核苷酸结合折叠定义的典型核心区域内。此外，许多LeuRS含有一个独特的多肽插入片段，由位于保守的KMSKS序列上游的约50个氨基酸组成。这个亮氨酸特异性结构域（LS结构域）的作用尚不清楚。我们推测该结构域可能对氨酰化过程中的底物识别和/或氨基酸编辑很重要。我们在大肠杆菌的亮氨酸特异性结构域内进行了一系列缺失突变和嵌合交换实验。我们的结果表明，亮氨酸特异性结构域对氨酰化至关重要，但对编辑活性不是必需的。动力学分析确定，LS结构域的缺失主要影响催化常数（kcat）。由于其靠近氨酰化活性位点，我们提出该结构域在氨基酸活化和/或tRNA氨酰化过程中与tRNA相互作用。尽管亮氨酸特异性结构域似乎对编辑复合体不重要，但它仍有可能辅助将tRNA从氨酰化复合体转移到编辑复合体的动态转运过程。
