M. Fraaije, W. V. van Berkel
Jul 18, 1997
Citations
0
Influential Citations
10
Citations
Journal
The Journal of Biological Chemistry
Abstract
The catalytic mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol by the covalent flavoprotein 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 reduction is rate determining. During anaerobic reduction 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 (ε364 nm = 46 mm −1cm−1). A similar transient species was observed upon anaerobic reduction by vanillyl alcohol. The rate of flavin reduction and synchronous intermediate 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. Oxidation 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 ternary complex mechanism in which the reduction rate is rate-limiting. It is proposed that, upon reduction, a binary complex is produced composed of thep-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme.