Fraaije M W, van den Heuvel R H, Roelofs J C, van Berkel W J
Department of Biochemistry, Wageningen Agricultural University, The Netherlands.
Eur J Biochem. 1998 May 1;253(3):712-9. doi: 10.1046/j.1432-1327.1998.2530712.x.
The kinetic mechanism of vanillyl-alcohol oxidase with 4-methylphenol, 4-ethylphenol, 4-propylphenol and their C alpha-deuterated analogs has been studied at pH 7.5 and 25 degrees 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 [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 rate of formation is too slow to be of catalytic relevance.
在pH 7.5和25℃条件下，研究了香草醇氧化酶与4-甲基苯酚、4-乙基苯酚、4-丙基苯酚及其α-氘代类似物的动力学机制。4-甲基苯酚的转化极其缓慢（0.005 s⁻¹），而在周转过程中酶主要处于还原形式。4-乙基苯酚和4-丙基苯酚很容易转化，而在周转过程中酶主要处于氧化形式。总体催化的氘动力学同位素效应在7至10之间，而黄素还原的固有氘动力学同位素效应超过9至10。对于所有三种4-烷基苯酚，黄素还原似乎是一个可逆过程，还原速率与逆反应速率处于相同范围。在香草醇氧化酶与4-乙基苯酚和4-丙基苯酚的还原半反应过程中，形成了一个瞬态中间体，其在330 nm处有最大吸光度。该中间体已初步鉴定为与还原酶结合的芳香底物的对醌甲基化物。得出的结论是，香草醇氧化酶与4-乙基苯酚和4-丙基苯酚的催化作用有利于有序的顺序结合机制，其中黄素还原速率决定周转速率，而还原酶-对醌甲基化物二元复合物与双氧快速反应。在香草醇氧化酶与4-甲基苯酚的反应过程中，一种荧光酶物种得以稳定。根据其光谱特征和晶体学数据[马特维，A.，弗拉伊耶，M. W.，莫扎雷利，A.，奥利维，L.，科达，A. & 范·伯克尔，W. J. H.（1997年）《结构》5，907 - 920]，提出该物种代表一种共价5 -（4'-羟基苄基）-FAD加合物。对于4-乙基苯酚和4-丙基苯酚，可能形成类似的N5黄素加合物，但它们的形成速率太慢，与催化无关。
