Spectroscopic and Computational Insights into the Mechanism of Cofactor Cobalt-Carbon Bond Homolysis by the Adenosylcobalamin-Dependent Enzyme Ethanolamine Ammonia-Lyase.

The adenosylcobalamin (AdoCbl)-dependent enzyme ethanolamine ammonia-lyase (EAL) catalyzes the conversion of ethanolamine to acetaldehyde and ammonia. As is the case for all AdoCbl-dependent isomerases, the catalytic cycle of EAL is initiated by homolytic cleavage of the cofactor's Co-C bond, producing Cocobalamin (CoCbl) and an adenosyl radical that serves to abstract a hydrogen atom from the substrate. Remarkably, in the presence of substrate, the rate of Co-C bond homolysis of enzyme-bound AdoCbl is increased by 12 orders of magnitude. For Class I AdoCbl-dependent isomerases, an important contribution to this rate acceleration stems from a stabilization of the CoCbl posthomolysis product by the axially coordinated histidine residue that displaces the pendant base from the Co ion upon AdoCbl binding to these enzymes. However, EAL and other Class II isomerases bind AdoCbl in the so-called "base-on" conformation and must therefore employ a different mechanism of Co-C bond activation. In the present study, we have used a combined spectroscopic and computational approach to probe the conformational changes and enzyme/cofactor/substrate interactions that contribute to the rate acceleration of Co-C bond homolysis in EAL. Spectroscopic data of AdoCbl and CoCbl show minimal perturbations upon cofactor binding to EAL in both the absence and presence of substrate. Structural models of free and EAL-bound AdoCbl were constructed using molecular dynamics and quantum mechanics/molecular mechanics computations. By carrying out relaxed potential energy scans for Co-C bond cleavage of free and EAL-bound AdoCbl, we identified key cofactor/enzyme interactions that contribute to the Co-C bond activation by EAL and obtained Co-C bond dissociation energies that agree well with published experimental data.