Hothi Parvinder, Hay Sam, Roujeinikova Anna, Sutcliffe Michael J, Lee Michael, Leys David, Cullis Paul M, Scrutton Nigel S
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Chembiochem. 2008 Nov 24;9(17):2839-45. doi: 10.1002/cbic.200800408.
Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme-substrate complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.
定量构效关系被广泛用于探究醌蛋白酶催化的C-H键断裂。然而，我们最近发现，对取代苄胺是醌蛋白芳香胺脱氢酶（AADH）较差的反应性探针，因为在C-H键断裂之前，酶-底物复合物需要发生结构变化。这种重排部分限制了反应速率，导致对取代苄胺的动力学同位素效应降低。在此，我们报告了AADH与对取代苯乙胺的反应性（驱动力）研究，对于后者，伴随C-H/C-(2)H键断裂的动力学同位素效应（约为16）高于半经典极限。我们表明键断裂是通过量子隧穿发生的，并且在氢隧穿的环境耦合框架内，对取代基的存在对快速促进运动的明显需求提出了更高要求。用苯乙胺或甲氧基苯乙胺浸泡的AADH晶体结构表明，对取代苄胺所确定的结构变化不应限制与对取代苯乙胺的反应。这与用对取代苯乙胺测得的升高的动力学同位素效应一致。我们发现质子转移的速率常数与反应性C-H键的键解离能之间存在良好的相关性，这与受类Marcus隧穿机制限制的速率一致。随着驱动力增大，质子转移速率增加，而Marcus活化能减小。这是首次使用一系列相关底物对酶中氢隧穿的驱动力扰动进行的实验报告。我们的研究为AADH中的质子隧穿提供了进一步的支持。
