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 Mechanism ² Parvinder Hothi, ‡ Michael Lee, § Paul M. Cullis, § David Leys, ‡ and Nigel S. Scrutton* ,‡ Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, UniVersity of Manchester, 131 Princess Street, Manchester, M1 7ND, U.K., and Department of Chemistry, UniVersity of Leicester, UniVersity Road, Leicester, LE1 7RH, U.K. ReceiVed August 21, 2007; ReVised Manuscript ReceiVed October 5, 2007 ABSTRACT: Para-substituted benzylamines are poor reactivity probes for structure-reactivity studies with TTQ-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 reactivity probes 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 small subunit of AADH catalyzes the oxidative deamination of a variety of amine substrates. Observed rate constants are second order with respect to substrate and inhibitor (phenylhydrazine) concentration. Kinetic studies with para-substituted benzylamines and their dideuterated counterparts reveal KIEs (>6) larger than 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 the native enzyme. This structural reorganization requires motions from the side chains of adjacent residues (which are absent in the isolated small subunit). The position of PheR97 in particular is responsible for the conformational gating (and hence deflated KIEs) observed with para-substituted benzylamines in the native enzyme. Hammett plots for the small subunit exhibit a strong correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenethylamines, unlike native AADH for which a poor correlation is observed. TTQ reduction in the isolated subunit is enhanced by electron withdrawing substituents, contrary to structure-reactivity studies reported for synthetic TTQ model compounds in which rate constants are enhanced by electron donating substituents. We infer that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated small subunit. This is attributed to the absence of structural rearrangement prior to H-transfer that limits the rate of TTQ reduction by para-substituted benzylamines in native enzyme. Tryptophan tryptophylquinone (TTQ 1 ) is a novel amino acid-derived cofactor found in the bacterial dehydrogenases aromatic amine dehydrogenase (AADH) and methylamine dehydrogenase (MADH) (1-4). TTQ is formed by post- translational modification of two gene-encoded tryptophan residues (5), and is tightly associated in the enzyme matrix through an amide linkage making it difficult to isolate the intact cofactor from native enzymes (1-6). Both TTQ- dependent enzymes adopt an R 2  2 heterotetrameric structure (R, 40 kDa; TTQ-containing , 14 kDa), and catalyze the oxidative deamination of a wide range of amines to their corresponding aldehydes and ammonia (2, 3). In the reductive half-reaction, two electrons released upon substrate oxidation are transferred to the TTQ cofactor. In the oxidative half- reaction, the TTQ is reoxidized by electron transfer to the type I blue copper proteins azurin (AADH) or amicyanin (MADH), respectively (2, 3). H-transfer in the reductive half-reaction of both TTQ- dependent dehydrogenases is consistent with full tunneling models of H-transfer and not the Bell tunneling correction model of semiclassical transition state theory (7-10). The mechanism of the reductive half-reaction of AADH is complex, involving several proton transfers followed by hydrolysis of the Schiff base-reduced TTQ adduct to a S-carbinolamine TTQ-adduct (Figure 1). With tryptamine substrate, H-tunneling occurs predominately to the O2 carboxylate of the active site base (Asp128) and is promoted by a short-range motion that modulates the proton-acceptor distance (9-11). In the oxidative half-reaction, hydrolysis of the product Schiff base produces the aminoquinol intermediate, which is converted back to the oxidized enzyme by two consecutive electron transfers to azurin (12). In native AADH, structural reorganization of the substrate-reduced ² The work was funded by the UK Biotechnology and Biological Sciences Research Council. N.S.S. is a BBSRC Professorial Research Fellow. * Correspondence to this author. Tel: +44 161 306 5152. Fax: +44 161 306 8918. E-mail nigel.scrutton@manchester.ac.uk. ‡ University of Manchester. § University of Leicester. 1 Abbreviations: AADH, aromatic amine dehydrogenase; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; KIE, kinetic iso- tope effect; MADH, methylamine dehydrogenase; QSARs, quantitative structure-activity relationships; TTQ, tryptophan tryptophylquinone; σp, electronic parameter for substituents in the para position; F, field/ inductive parameter; R, resonance parameter. 183 Biochemistry 2008, 47, 183-194 10.1021/bi701690u CCC: $40.75 © 2008 American Chemical Society Published on Web 12/05/2007