Catalytic Mechanism of the Oxidative Demethylation of 4-(Methoxymethyl)phenol by Vanillyl-Alcohol Oxidase

Abstract

The catalytic mechanism for the oxidative demethyla- tion of 4-(methoxymethyl)phenol by the covalent fla- voprotein vanillyl-alcohol oxidase was studied. Using H2 18O, it was found that the carbonylic oxygen atom from the product 4-hydroxybenzaldehyde originates from a water molecule. Oxidation of vanillyl alcohol did not result in any incorporation of 18O. Enzyme-monitored turnover experiments revealed that for both substrates a process involving flavin re- duction is rate determining. During anaerobic reduc- tion of vanillyl-alcohol oxidase by 4-(methoxymethyl)- phenol, a relatively stable spectral intermediate is formed. Deconvolution of its spectral characteristics showed a typical pH-independent absorption maximum at 364 nm ( ? 364 nm ? 46 mM?1 cm?1). A similar transient species was observed upon anaerobic reduction by vanillyl alcohol. The rate of flavin reduction and synchronous interme- diate formation by 4-(methoxymethyl)phenol is 3.3 s?1 and is fast enough to account for turnover (3.1 s?1). The anaerobic decay of the intermediate was too slow (0.01 s?1) to be of catalytical relevance. The reduced binary complex is rapidly reoxidized (1.5 ? 105 M?1 s?1) and is accompanied with formation and release of product. Ox- idation of free-reduced enzyme is an even faster process (3.1 ? 105 M?1 s?1). The kinetic data for the oxidative demethylation of 4-(methoxymethyl)phenol are in accordance with a ter- nary complex mechanism in which the reduction rate is rate-limiting. It is proposed that, upon reduction, a bi- nary complex is produced composed of the p-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme. Vanillyl-alcohol