Molecular orbital studies of enzyme mechanisms. II. Catalytic oxidation of alcohols by liver alcohol dehydrogenase

Semiempirical (AM1) molecular orbital theory has been used to investigate the oxidation of alcohols at the active site of liver alcohol dehydrogenase (LADH). The model active site consists of a zinc dication coordinated to two methyl‐mercaptans (Cys‐46, Cys‐176), an imidazole (His‐67), and a water. An imidazole (His‐51) hydrogen bonded to a hydroxy‐acetate (Ser‐48) forms the remote base. AM1 calculations that address the two distinct steps in the catalytic mechanism of ethanol oxidation by LADH are reported. These two steps are: (1) the deprotonation of ethanol by imidazole (His‐51) via hydrogen‐bonded hydroxy‐acetate (Ser‐48), creating a proton relay system; and (2) the rate‐limiting hydride transfer step from ethanol C1 to nicotinamide adenine dinucleotide (NAD+), leading to product formation. Detailed calculations have been used to resolve the unsolved problems of mechanisms that have been suggested on the basis of kinetic data and crystal structures of several LADH complexes. We investigated two possible mechanisms for the deprotonation of ethanol, by zinc‐bound OH− and by direct deprotonation of zinc‐bound ethanol by imidazole via hydroxyacetate (Ser‐48). Our calculations show that there is no need for LADH to activate a water molecule at the active site as in many other zinc enzymes. This result agrees with experimental evidence. Our calculations also indicate that substrates are bound in an inner‐sphere‐pentacoordinated complex to the active site zincion. In this case, spectroscopic investigations agree with our results but crystallographic data do not. The highest activation energy is found for the hydride transfer, in agreement with the experiment. Finally, we proposed an alternative mechanism for the mode of action of LADH based upon our results. © 1993 John Wiley & Sons, Inc.