Increasing complexity models for describing the generation of substrate radicals at the active site of ethanolamine ammonia-lyase/B12

Ethanolamine ammonia-lyase (EAL) is an adenosylcobalamin (AdoCbl, a B12 coenzyme) dependent enzyme expressed in enteric bacteria, which catalyzes conversion of ethanolamine in ammonia and acetaldehyde under anaerobic conditions. The most accepted reaction mechanism includes five steps: (a) 5-deoxyadenosyl radical (Ado) generation by homolytic cleavage of a Co–C bond in AdoCbl; (b) substrate activation by Ado H-abstraction from C1; (c) 1,2-NH2 migration in substrate’s radical; (d) Ado regeneration; (e) NH3 elimination from substrate leaving acetaldehyde as final product. Steps a–c have been the subject of both empirical studies and computational modeling performed at the ab initio and DFT levels, with different representations of the protein environment, attempting to explain the observed kinetics by total/partial protonation or by “push–pull” catalysis assisted by two amino acid residues. In such models, a drastic reduction of the reactive system and of the influence of the enzyme environment was introduced, the latter mainly due to the lack of detailed data on the 3D structure of EAL (note elucidated until 2007) and the nature of the active site (for which a structure of the substrate-EAL complex in presence of adeninylpentylcobalamin has been very recently reported, shedding light on its structural features). e assess at the PCM//UB3LYP/6-31G(d,p) level how using a complete molecular description of both reactants in step b and introducing the polarizing effect of the protein/cofactor environment (mimicked through a continuum model with ε = 10) impacts on the nature and energetics of the intermediate complexes (ICs) and transition states (TSs) under three catalytic scenarios (total, partial, and no protonation). Our results show that a concerted asynchronous mechanism involving distonic radical species is at play, passing through quasi-linear early TSs displaying a C1–H cleavage slightly more advanced than formation of the new H–C bond in Ado. Whereas the enzyme’s polarizing environment differentially stabilizes the TS lowering the barrier by ∼2.4–2.8 kcal/mol, a reduction of 1.4 kcal/mol in Δ G 298 ≠ is the outcome of substrate’s partial/total protonation, without a clear distinction among them, in opposition to what previously shown based upon more simplified models.