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|Title:||Isotope effects reveal that p-substituted bezylamines are poor reactivity probes of quinoprotein mechanism for aromatic amine dehydrogenase|
Abu Khadra, K.
Cullis, P. M.
Scrutton, N. S.
|Citation:||Biochemistry, 2007, 46 (32), pp.9250-9259|
|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.|
|Appears in Collections:||Published Articles, Dept. of Chemistry|
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