Isotope Effects Reveal That Para-Substituted Benzylamines Are Poor Reactivity Probes of the Quinoprotein Mechanism for Aromatic Amine Dehydrogenase
详细信息 查看全文
- 作者：Parvinder Hothi ; Anna Roujeinikova ; Khalid Abu Khadra ; Michael Lee ; Paul Cullis ; David Leys ; Nigel S. Scrutton
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：August 14, 2007
- 年：2007
- 卷：46
- 期：32
- 页码：9250 - 9259
- 全文大小：423K
- 年卷期：v.46,no.32(August 14, 2007)
- ISSN：1520-4995
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.