Studies on the mechanism of alcohol oxidase

Abstract

The flavoprotein alcohol oxidase from the yeast Candida boidinii catalyzes the oxidation of primary alcohols to aidehydes with transfer of the electrons to molecular oxygen to form hydrogen peroxide. The mechanism of alcohol oxidase with beta substituted ethanois as substrates has been examined using kinetic isotope effects, structure-reactivity correlations, and pH effects. Initial velocity line patterns for ethanol and bromoethanol as substrates for alcohol oxidase showed parallel lines, consistent with an irreversible step between the binding of the alcohol and oxygen, whereas methoxyethanol, fluoroethanol, chloroethanol, iodoethanol, and trifluoroethanol showed intersecting line patterns, consistent with a reversible step between the binding of the alcohol and oxygen. The effects of substituents in beta substituted ethanois on V/K values showed a good correlation with the a, value of the substituent after correction for steric, and hydrophobic effects, with a p value of-1.24. This value is inconsistent with a carbanion intermediate. Primary and solvent deuterium isotope effects were determined on the V/K values for several beta substituted ethanols. As the primary deuterium isotope effect increased, the solvent isotope effect decreased. The observed D(V/K)EtOH values in H2O (1 .54 ︢0.14) and D20 (1.51 ︢0.19) were similar, consistent with a concerted mechanism. The above studies are consistent with an asynchronous concerted mechanism in which the OH bond cleavage is further advanced than carbon hydrogen bond cleavage. With methanol, ethanol, and trifluoroethanol as substrates for alcohol oxidase, a single ionizable group with a pKa value of 8.3 must be deprotonated for binding and catalysis. This residue is proposed to be histidine. The D(V/K)EtOH values at low pH (1.62 ︢0.12) and high pH (1.54 ︢ 0.14)were similar, consistent with ethanol not being a sticky substrate.