Substituted Alcohols as Mechanistic Probes of Alcohol Oxidase

The flavoprotein alcohol oxidase catalyzes the oxidation of primary and secondary alcohols to aldehydes, transferring the electrons to oxygen to form hydrogen peroxide. In order to probe the mechanism of oxidation, para-substituted benzyl alcohols and β-substituted ethyl alcohols have been characterized as substrates. With benzyl alcohol, methanol, and ethanol, a single group on the protein with a pKa value of about 0.3 must be deprotonated for productive binding. The effects of substituents in para-substituted benzyl alcohols on the V/K value show a good correlation with the σ− value of the substituent after correction for hydrophobic and steric contributions, with a ρ Value of 1.9. The effects of substituents in β-substituted ethyl alcohols show a good correlation with the σI value of the substituent after correction for steric and hydrophobic effects, with a ρ Value of −1.2. Solvent and primary deuterium kinetic isotope effects were determined on the V/K values for several alcohols. Substrates showed either a significant primary effect or a significant solvent isotope effect. The results are interpreted in terms of an asynchronous concerted transition state in which OH bond cleavage has occurred to a greater extent than CH bond cleavage.