Murakawa Takeshi, Okajima Toshihide, Kuroda Shun'ichi, Nakamoto Takuya, Taki Masayasu, Yamamoto Yukio, Hayashi Hideyuki, Tanizawa Katsuyuki
Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Biochem Biophys Res Commun. 2006 Apr 7;342(2):414-23. doi: 10.1016/j.bbrc.2006.01.150. Epub 2006 Feb 8.
A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate alpha-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the alpha-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reaction of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics.
一个决定性影响铜胺氧化酶催化效率的关键步骤是由一个保守的天冬氨酸残基对底物α-质子进行立体特异性提取。我们使用一种细菌酶和立体特异性氘标记的底物2-苯乙胺和酪胺，通过预稳态动力学分析了这一步骤。在α-质子提取步骤中，2-苯乙胺观察到一个小的且与温度相关的动力学同位素效应（KIE），而酪胺观察到一个大的且与温度无关的KIE，这表明该步骤是由量子力学氢隧穿而非经典过渡态机制驱动的。此外，在缺乏关键天冬氨酸的突变酶反应中获得了接近过渡态值的阿累尼乌斯型指前因子比。这些结果为酶增强氢隧穿提供了有力证据。反应中间体的X射线晶体结构揭示了底物远端部分结合模式的微小差异，这将通过主动或被动动力学调节氢隧穿过程。
