Catalysis by the Isolated Tryptophan Tryptophylquinone-Containing Subunit of Aromatic Amine Dehydrogenase Is Distinct from Native Enzyme and Synthetic Model Compounds and Allows Further Probing of TTQ

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• 作者：Parvinder Hothi ; Michael Lee ; Paul M. Cullis ; David Leys ; Nigel S. Scrutton
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：January 8, 2008
• 年：2008
• 卷：47
• 期：1
• 页码：183 - 194
• 全文大小：415K
• 年卷期：v.47,no.1(January 8, 2008)
• ISSN：1520-4995

文摘

Para-substituted benzylamines are poor reactivity probes for structure-reactivity studies withTTQ-dependent aromatic amine dehydrogenase (AADH). In this study, we combine kinetic isotope effects(KIEs) with structure-reactivity studies to show that para-substituted benzylamines are good reactivityprobes of TTQ mechanism with the isolated TTQ-containing subunit of AADH. Contrary to the TTQ-containing subunit of methylamine dehydrogenase (MADH), which is catalytically inactive, the smallsubunit of AADH catalyzes the oxidative deamination of a variety of amine substrates. Observed rateconstants are second order with respect to substrate and inhibitor (phenylhydrazine) concentration. Kineticstudies with para-substituted benzylamines and their dideuterated counterparts reveal KIEs (>6) largerthan those observed with native AADH (KIEs ~ unity). This is attributed to formation of the benzylamine-derived iminoquinone requiring structural rearrangement of the benzyl side chain in the active site of thenative enzyme. This structural reorganization requires motions from the side chains of adjacent residues(which are absent in the isolated small subunit). The position of Phe97 in particular is responsible forthe conformational gating (and hence deflated KIEs) observed with para-substituted benzylamines in thenative enzyme. Hammett plots for the small subunit exhibit a strong correlation of structure-reactivitydata with electronic substituent effects for para-substituted benzylamines and phenethylamines, unlikenative AADH for which a poor correlation is observed. TTQ reduction in the isolated subunit is enhancedby electron withdrawing substituents, contrary to structure-reactivity studies reported for synthetic TTQmodel compounds in which rate constants are enhanced by electron donating substituents. We infer thatpara-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated smallsubunit. This is attributed to the absence of structural rearrangement prior to H-transfer that limits therate of TTQ reduction by para-substituted benzylamines in native enzyme.