Hothi Parvinder, Roujeinikova Anna, Khadra Khalid Abu, Lee Michael, Cullis Paul, Leys David, Scrutton Nigel S
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, UK.
Biochemistry. 2007 Aug 14;46(32):9250-9. doi: 10.1021/bi7007239. Epub 2007 Jul 18.
Structure-activity correlations have been employed previously in the mechanistic interpretation of TTQ-dependent amine dehydrogenases using a series of para-substituted benzylamines. However, by combining the use of kinetic isotope effects (KIEs) and crystallographic analysis, in conjunction with structure-reactivity correlation studies, we show that para-substituted benzylamines are poor reactivity probes for TTQ-dependent aromatic amine dehydrogenase (AADH). Stopped-flow kinetic studies of the reductive half-reaction, with para-substituted benzylamines and their dideuterated counterparts, demonstrate that C-H or C-D bond breakage is not fully rate limiting (KIEs approximately unity). Contrary to previous reports, Hammett plots exhibit a poor correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenylethylamines. Crystallographic studies of enzyme-substrate complexes reveal that the observed structure-reactivity correlations are not attributed to distinct binding modes for para-substituted benzylamines in the active site, although two binding sites for p-nitrobenzylamine are identified. We identify structural rearrangements, prior to the H-transfer step, which are likely to limit the rate of TTQ reduction by benzylamines. This work emphasizes (i) the need for caution when applying structure-activity correlations to enzyme-catalyzed reactions and (ii) the added benefit of using both isotope effects and structural analysis, in conjunction with structure-reactivity relationships, to study chemical steps in enzyme reaction cycles.
结构-活性关系先前已被用于通过一系列对位取代苄胺对依赖TTQ的胺脱氢酶进行机理阐释。然而，通过结合动力学同位素效应（KIEs）的应用和晶体学分析，并与结构-反应性相关研究相结合，我们发现对位取代苄胺对于依赖TTQ的芳香胺脱氢酶（AADH）而言是较差的反应性探针。使用对位取代苄胺及其双氘代对应物对还原半反应进行的停流动力学研究表明，C-H或C-D键的断裂并非完全是速率限制因素（KIEs约为1）。与先前的报道相反，哈米特图显示对位取代苄胺和苯乙胺的结构-反应性数据与电子取代基效应之间的相关性较差。酶-底物复合物的晶体学研究表明，尽管确定了对硝基苄胺的两个结合位点，但观察到的结构-反应性相关性并非归因于对位取代苄胺在活性位点的不同结合模式。我们确定了在氢转移步骤之前的结构重排，这可能会限制苄胺对TTQ的还原速率。这项工作强调了（i）在将结构-活性关系应用于酶催化反应时需要谨慎，以及（ii）结合同位素效应和结构分析，并与结构-反应性关系相结合来研究酶反应循环中的化学步骤所带来的额外益处。
