Yukawa Ayako, Iino Ryota, Watanabe Rikiya, Hayashi Shigehiko, Noji Hiroyuki
Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo , Tokyo 113-8656, Japan.
Biochemistry. 2015 Jan 20;54(2):472-80. doi: 10.1021/bi501138b. Epub 2014 Dec 30.
A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F1-ATPase (F1), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F1 in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH2 unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F1. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high "chemical" resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics.
一种具有催化重要性的精氨酸，称为精氨酸指，在许多酶中发挥作用，通过核苷酸三磷酸的酶促水解来调节其功能。F1 - ATP合酶（F1）是一种旋转马达蛋白，拥有精氨酸指，可催化三磷酸腺苷（ATP）的水解，以实现高效的化学机械能转换。在本研究中，我们通过单分子测量研究了F1的一个突变体的精氨酸指催化作用，该突变体中精氨酸指被赖氨酸类似物2,7 - 二氨基庚酸（Lyk）这种非天然氨基酸取代。使用Lyk，其侧链通过一个CH2单元延长，使得其到胺末端氮的链长与精氨酸的链长相等，这使我们能够解析精氨酸指催化中的关键化学因素，即链长匹配和末端基团的化学性质。通过单分子观察进行的速率测量表明，侧链长度的链长匹配并非精氨酸指催化ATP水解反应步骤的唯一要求，这表明精氨酸指催化中末端胍基化学性质的至关重要性。另一方面，Lyk突变阻止了赖氨酸突变体中观察到的严重的ADP抑制状态的形成，甚至与野生型F1相比，提高了对抑制的规避能力。本研究表明，非天然氨基酸的掺入可以以其高“化学”分辨率广泛扩展生物化学方法，用于阐明蛋白质功能的分子机制并赋予新特性。
