Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
J Am Chem Soc. 2012 Dec 19;134(50):20254-7. doi: 10.1021/ja309646s. Epub 2012 Dec 4.
Using high-speed scanning atomic force microscopy, we directly observed single-molecular enzymatic elongation of hyaluronan polymer chains at intervals of 10 s on a mica or lipid bilayer surface, on which Pasteurella multocida hyaluronic acid synthase (pmHAS) was immobilized. The reaction was started by the addition of both UDP-glucuronic acid and UDP-N-acetylglucosamine monomers. The average catalytic elongation rate constant (k(cat)) was found to be 1.8 mer s(-1) from one active enzyme physically adsorbed on a mica surface. When pmHAS was immobilized by inserting its hydrophobic tail part into lipid bilayers, most of the enzymes retained their activity, and the k(cat) values were found to be in the range 1-10 mer s(-1) for 29 enzymes (average was k(cat) = 2-4 mer s(-1)). These k(cat) values were lowest level of k(cat) = 1-100 s(-1) obtained in bulk solution by radioisotope methods.
利用高速扫描原子力显微镜，我们直接观察到在云母或脂质双层表面上固定的多杀巴斯德氏菌透明质酸合酶（pmHAS）上，每隔 10 秒就会有透明质酸聚合物链的单分子酶促延伸。反应通过添加 UDP-葡萄糖醛酸和 UDP-N-乙酰葡萄糖胺单体来启动。从物理吸附在云母表面上的一个活性酶中发现，平均催化延伸速率常数（kcat）为 1.8 个 mer s-1。当 pmHAS 通过插入其疏水尾部部分固定在脂质双层中时，大多数酶保留其活性，并且发现 29 种酶的 kcat 值在 1-10 mer s-1 范围内（平均值为 kcat = 2-4 mer s-1）。这些 kcat 值是通过放射性同位素方法在溶液中获得的 1-100 s-1 最低 kcat 值。
