Catalytic Mechanism of Human Aldehyde Oxidase

The
mechanism of oxidation of N-heterocycle phthalazine
to phthalazin-1­(2H)-one and its associated free energy
profile, catalyzed by human aldehyde oxidase (hAOX1), was studied
in atomistic detail using QM/MM methodologies. The studied reaction
was found to involve three sequential steps: (i) protonation of the
substrate’s N2 atom by Lys893, (ii) nucleophilic attack of
the hydroxyl group of the molybdenum cofactor (Moco) to the substrate,
and (iii) hydride transfer from the substrate to the sulfur atom of
the Moco. The free energy profile that was calculated revealed that
the rate-limiting step corresponds to hydride transfer. It was also
found that Lys893 plays a relevant role in the reaction, being important
not only for the anchorage of the substrate close to the Moco, but
also in the catalytic reaction. The variations of the oxidation state
of the molybdenum ion throughout the catalytic cycle were examined
too. We found out that during the displacement of the products away
from the Moco, the transfer of electrons from the catalytic site to
the FAD site was proton-coupled. As a consequence, the most favorable
and fastest pathway for the enzyme to complete its catalytic cycle
was that with MoV and a deprotonated SH ligand of the Moco
with the FAD molecule converted to its semiquinone form, FADH•.