Department of Engineering Science, Bioscience and Technology Program, The Graduate School of Informatics and Engineering, The University of Electro-Communications (UEC), 7-5-1 Chofugaoka, Chofu, Tokyo 182-8585, Japan.
FEBS Open Bio. 2013 Jun 11;3:252-5. doi: 10.1016/j.fob.2013.06.001. Print 2013.
The introduction of non-natural amino acids at the N-terminus of peptides/proteins using leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is a useful technique for protein engineering. To accelerate the chemoenzymatic reaction, here we systematically optimized the N-terminal penultimate residue of the acceptor peptide. Positively charged, small, or hydrophilic amino acids at this position show positive effects for the reaction. Kinetic analysis of peptides possessing different penultimate residues suggests that the side chain of the residue affects peptide-binding affinity towards the L/F-transferase. These findings also provide biological insight into the effect of the penultimate amino acid on substrate specificity of natural proteins to be degraded via the N-end rule pathway.
使用亮氨酰/苯丙氨酰-tRNA 蛋白转移酶（L/F-转移酶）在肽/蛋白质的 N 端引入非天然氨基酸是一种有用的蛋白质工程技术。为了加速化学酶反应，我们在这里系统地优化了受体肽的 N 端倒数第二位残基。该位置带正电荷、小或亲水的氨基酸对反应有积极影响。具有不同倒数第二位残基的肽的动力学分析表明，残基的侧链影响肽与 L/F-转移酶的结合亲和力。这些发现还为 N 端规则途径降解的天然蛋白质的底物特异性的最后一个氨基酸的影响提供了生物学上的深入了解。
