Interaction of two arginine residues in lactate oxidase with the enzyme flavin: conversion of FMN to 8-formyl-FMN.
Interaction of two arginine residues in lactate oxidase with the enzyme flavin: conversion of FMN to 8-formyl-FMN.
复制标题
乳酸氧化酶中两个精氨酸残基与黄素酶的相互作用：FMN 转化为 8-甲酰基-FMN。
DOI：
10.1073/pnas.250472297
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发表时间：
2000
影响因子：
11.1
通讯作者：
Massey,V
中科院分区：
文献类型：
--
作者：
Yorita,K;Matsuoka,T;Misaki,H;Massey,V
两个精氨酸残基 Arg-181 和 Arg-268 在已知的催化 α-羟基酸氧化的含 FMN 酶家族中是保守的。在来自草绿色气球菌的乳酸氧化酶中，这些残基已在两个单突变和双突变形式中变成赖氨酸。此外，Arg-181已被蛋氨酸取代，以确定去除残留物上正电荷的效果。报告了这些替代物对动力学和热力学性质的影响。对于所有突变形式，对还原黄素与氧的反应性只有很小的影响。另一方面，氧化黄素乳酸丁酯的还原效率大大降低，特别是对于R268K突变体形式。结果证明了两个精氨酸残基在底物结合及其与黄素相互作用中的重要性，并且与之前的假设一致，即它们在底物脱氢的过渡态中也起到电荷中和的作用。 Arg-268被赖氨酸取代还导致FMN的8-CH3-取代基缓慢转化为8-甲酰基-FMN，其仍然与酶紧密结合，并且具有与FMN酶显着不同的物理和化学性质。
Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of α-hydroxyacids. In the lactate oxidase fromAerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin byl-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.