Kinetic mechanism of vanillyl-alcohol oxidase with short-chain 4-alkylphenols

The kinetic mechanism of vanillyl-alcohol oxidase with 4-methylphenol, 4-ethylphenol, 4-propylphenol and their Cα-deuterated analogs has been studied at pH 7.5 and 25°C. Conversion of 4-methylphenol is extremely slow (0.005 s-1) while the enzyme is largely in the reduced form during turnover. 4-Ethylphenol and 4-propylphenol are readily converted while the enzyme is mainly in the oxidized form during turnover. The deuterium kinetic isotope effect for overall catalysis ranges between 7-10 whereas the intrinsic deuterium kinetic isotope effect for flavin reduction ranges over 9-10. With all three 4-alkylphenols, flavin reduction appeared to be a reversible process with the rate of reduction being in the same range as the rate for the reverse reaction. During the reductive half-reaction of vanillyl-alcohol oxidase with 4-ethylphenol and 4-propylphenol, a transient intermediate is formed with an absorbance maximum at 330 nm. This intermediate has been tentatively identified as the p-quinone methide of the aromatic substrate in complex with reduced enzyme. It is concluded that vanillyl-alcohol oxidase catalysis with 4-ethylphenol and 4-propylphenol favors an ordered sequential binding mechanism in which the rate of flavin reduction determines the turnover rate while the reduced enzyme-p-quinone methide binary complex rapidly reacts with dioxygen. During the reaction of vanillyl-alcohol oxidase with 4-methylphenol, a fluorescent enzyme species is stabilized. Based on its spectal characteristics and crystallographic data, it is proposed that this species represents a covalent 5-(4'-hydroxybenzyl)-FAD adduct. With 4-ethylphenol and 4-propylphenol, similar N5 flavin adducts may be formed but their rate of formation is too slow to be of catalytic relevance.