Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a p-quinone methide intermediate.
The catalytic mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol by the covalent flavoprotein vanillyl-alcohol oxidase was studied. Using H218O, 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 (epsilon364 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 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 x 10(5) 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 x 10(5) 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 the p-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme.
Fraaije
M W
MW
Department of Biochemistry, Agricultural University Wageningen, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
van Berkel
W J
WJ
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000693:Anaerobiosis; D002384:Catalysis; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes; D010407:Penicillium / Q000201:enzymology*; D010636:Phenols / Q000378:metabolism*; D011809:Quinones / Q000032:analysis*; D013053:Spectrophotometry; D013379:Substrate Specificity; D014867:Water
D010103:Oxygen Isotopes; D010636:Phenols; D011809:Quinones; D014867:Water; C107181:4-(methoxymethyl)phenol; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase