Sytina Olga A, Heyes Derren J, Hunter C Neil, Alexandre Maxime T, van Stokkum Ivo H M, van Grondelle Rienk, Groot Marie Louise
Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
Nature. 2008 Dec 18;456(7224):1001-4. doi: 10.1038/nature07354.
The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.
构象变化在解释酶的巨大催化能力中所起的作用，是当前生物学中最具挑战性的问题之一。尽管现在人们普遍认为酶通过短程和长程蛋白质运动来调节反应速率，但几乎不可能区分构象变化和催化作用。我们利用叶绿素生物合成酶NADPH：原叶绿素酸酯（Pchlide）氧化还原酶解决了这个问题，该酶催化一个涉及氢化物和质子转移的独特光驱动反应。在此我们报告，用激光脉冲预先激发酶 - 底物复合物会诱导活性位点形成更有利的构象，从而使氢化物和质子的偶联转移反应能够发生。这种效应在Pchlide激发态寿命期间触发，并在长时间尺度上持续存在，将酶转变为一种活性状态，其特征是催化中间体形成的速率和量子产率很高。吸收一个光子后在中红外区域相应的光谱变化揭示了酶中显著的构象变化，说明了酶结构中的灵活性和动力学对其功能的重要性。
