Computational studies of the mechanism for proton and hydride transfer in liver alcohol dehydrogenase

In this paper we present computational studies directed at elucidating the mechanism of the oxidation of benzyl alcohol by liver alcohol dehydrogenase (LADH). This enzyme reaction involves a hydride transfer from the alcohol substrate to the nicotinamide adenine dinucleotide coenzyme and a proton re lay that deprotonates the alcohol substrate. Electronic structure calculati ons at various levels of theory were performed on a 148-atom model of the a ctive site, and classical molecular dynamics simulations were performed on the entire solvated LADH dimer. These calculations support the hypothesis t hat alcohol deprotonation occurs prior to the hydride transfer step and tha t the alcohol deprotonation facilitates the hydride transfer by lowering th e barrier for hydride transfer. In this postulated mechanism, the alcohol d eprotonation leads to a zinc-bound alkoxide ion, and the subsequent hydride transfer leads to the benzaldehyde product. The calculations indicate that the zinc-bound alkoxide forms a strong hydrogen bond to Ser48 and that hyd ride transfer is accompanied by a weakening of this hydrogen bond. The resu lts also suggest that the barrier to hydride transfer is lowered by the ele ctrostatic interaction between the substrate alkoxide oxygen and the zinc c ounterion in the active site. The interaction of the alkoxide oxygen lone p air orbitals with the zinc competes with the formation of the double bond r equired for the aldehyde product, resulting in an earlier, more alcohol-lik e transition state and thus a lower activation energy barrier. In addition, the interaction between the alkoxide oxygen and the zinc restricts the dyn amical motion of the substrate, decreasing the average donor-acceptor dista nce for hydride transfer and hence lowering the activation energy barrier.