文摘
Structure-activity correlations have been employed previously in the mechanistic interpretationof TTQ-dependent amine dehydrogenases using a series of para-substituted benzylamines. However, bycombining the use of kinetic isotope effects (KIEs) and crystallographic analysis, in conjunction withstructure-reactivity correlation studies, we show that para-substituted benzylamines are poor reactivityprobes for TTQ-dependent aromatic amine dehydrogenase (AADH). Stopped-flow kinetic studies of thereductive half-reaction, with para-substituted benzylamines and their dideuterated counterparts, demonstratethat 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 effectsfor para-substituted benzylamines and phenylethylamines. Crystallographic studies of enzyme-substratecomplexes reveal that the observed structure-reactivity correlations are not attributed to distinct bindingmodes 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, whichare likely to limit the rate of TTQ reduction by benzylamines. This work emphasizes (i) the need forcaution when applying structure-activity correlations to enzyme-catalyzed reactions and (ii) the addedbenefit of using both isotope effects and structural analysis, in conjunction with structure-reactivityrelationships, to study chemical steps in enzyme reaction cycles.
