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Isotope effects reveal that p-substituted bezylamines are poor reactivity probes of quinoprotein mechanism for aromatic amine dehydrogenase

By P. Hothi, A. Roujeinikova, K. Abu Khadra, M. Lee, P. M. Cullis, D. Leys and N. S. Scrutton

Abstract

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 ∼ 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

Year: 2007
DOI identifier: 10.1021/bi7007239
OAI identifier: oai:lra.le.ac.uk:2381/581
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