Isotope Effects Reveal That Para-Substituted Benzylamines Are Poor Reactivity Probes of the Quinoprotein Mechanism for Aromatic Amine Dehydrogenase^,

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 approximately 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.

Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

“…Recent studies on tryptophan tryptophylquinone-dependent AADH also show that caution should be taken when using QSAR studies to analyze the chemistry of enzyme mechanism. The use of isotope effects in the case of AADH reveals that p-substituted benzylamines are poor reactivity probes, as proton abstraction is not fully rate-limiting (18). Our isotope effect studies with benzylamine suggest a complex kinetic mechanism for VAP-1, consistent with mechanistic schemes for the reductive half-reactions of related CAOs.…”

“…p-Bromophenylethylamine was from Fluorochem and p-methoxyphenylethylamine from Apollo Scientific Ltd. Perdeuterated phenylethylamine (C 6 D 5 CD 2 CD 2 NH 2 HCl, 99.6%) and horseradish peroxidase were from Sigma. Di-deuterated p-substituted phenylethylamines and d 2 -benzylamine were synthesized previously as described by Hothi et al (17,18). Monoclonal mouse anti-VAP-1 antibody was obtained from Bender Medsystems.…”

Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex

“…[9][10][11] Insight into the reaction cycle has come from detailed analysis of the crystal structures of reaction intermediates, [8,9,12] computational simulations of the reaction chemistry [8,13,14] and isotope analysis of the proton transfer step using fast reaction stopped-flow methods. [5,15,16] These studies have provided a detailed appreciation of the reaction chemistry, whilst emphasizing the importance of proton transfer by quantum tunnelling mechanisms (for example, with tryptamine substrate). Studies with other substrates (for example, p-substituted benzylamines) have revealed the need for structural reorganization prior to proton transfer.…”

“…Studies with other substrates (for example, p-substituted benzylamines) have revealed the need for structural reorganization prior to proton transfer. [5] This structural reorganization complicates analysis of the proton transfer step in studies of the temperature dependence of kinetic isotope effects (KIEs), which can be often used to infer tunnelling mechanisms. [17,18] In principle, studies of quantitative structure-activity relationships (QSAR) are useful for inferring mechanistic information, particularly with quinoprotein enzyme systems.…”

“…[1][2][3][4] However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme-substrate complex prior to CÀH bond breakage. [5] This A C H T U N G T R E N N U N G rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (~16) accompanying CÀH/CÀ 2 H bond breakage is elevated above the semi-classical limit.…”