KINETIC MECHANISM OF VANILLYL-ALCOHOL OXIDASE WITH SHORT-CHAIN 4-ALKYLPHENOLS

The kinetic mechanism of vanillyl-alcohoI oxidase with 4-methylphenol, 4-ethylphenol, di-propylphenol and their C alpha-deuterated analogs h as been studied at pH 7.5 and 25 degrees C. Conversion of 4-methylphen ol is extremely slow (0.005 s(-1)) while the enzyme is largely in the reduced form during turnover. 4-Ethylphenol and 4-propylphenol are rea dily converted while the enzyme is mainly in the oxidized form during turnover. The deuterium kinetic isotope effect for overall catalysis r anges between 7-10 whereas the intrinsic deuterium kinetic isotope eff ect for flavin reduction ranges over 9-10. With all three 4-alkylpheno ls, flavin reduction appeared to be a reversible process with the rate of reduction being in the same range as the rate for the reverse reac tion. During the reductive half-reaction of vanillyl-alcohol oxidase w ith 4-ethylphenol and 4-propylphenol, a transient intermediate is form ed with an absorbance maximum at 330 nm. This intermediate has been te ntatively identified as the p-quinone methide of the aromatic substrat e in complex with reduced enzyme. It is concluded that vanillyl-alcoho l oxidase catalysis with 4-ethylphenol and 4-propylphenol favors an or dered sequential binding mechanism in which the rate of flavin reducti on determines the turnover rate while the reduced enzyme-p-quinone met hide 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 crysta llographic data [Mattevi. A., Fraaije, M. W., Mozzarelli, A., Olivi, L ., Coda. A. & van Berkel, W. J. H. (1997) Structure 5, 907-920], 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 rare of formation is too slow to be of catalytic relevance.