Mechanisms of ammonia activation and ammonium ion inhibition of quinoprotein methanol dehydrogenase: A computational approach

Abstract

The mechanism of methanol oxidation by quinoprotein methanol dehydrogenase (MDH.PQQ) in combination with methanol (MDH.PQQ.methanol) involves Glu-171--CO2(-) general base removal of the hydroxyl proton of methanol in concert with hydride equivalent transfer to the >C5=O quinone carbon of pyrroloquinoline quinone (PQQ) and rearrangement to hydroquinone (PQQH2) with release of formaldehyde. Molecular dynamics (MD) studies of the structures of MDH.PQQ.methanol in the presence of activator NH3 and inhibitor NH4(+) have been carried out. In the MD structure of MDH.PQQ.methanol.NH3, the hydrated NH3 resides at a distance of approximately 24 A away from methanol and the ortho-quinone portion of PQQ. As such, influence of NH3 on the oxidation reaction is not probable. We find that NH4(+) competes with the substrate by hydrogen-bonding to Glu-171CO2(-) such that the MDH.PQQ.methanol.NH4(+) complex is not reactive. Ammonia readily forms imines with quinone. Imines are present in solution as neutral (>C5=NH) and protonated (>C5=NH2(+)) species. MD simulations establish that the >C5=NH2(+) derivative of MDH.PQQ(NH2(+).methanol structure is unreactive because of the nonproductive means of methanol binding. The structure obtained by the MD simulations with the neutral >C5=NH imine of MDH.PQQ(NH).methanol structure is similar to the reactive MDH.PQQ.methanol complex. This active site geometry allows for catalysis of hydride equivalent transfer to the >C5=NH of PQQ(NH) by concerted Glu-171CO(2)(-) general-base removal of the H-OCH3 proton and Arg-324H+ general-acid proton transfer to the imine nitrogen. Enzyme-bound <C5(H)NH2 derivative of PQQ [PQQ(NH)] and CH(2)O product are formed.