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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >On the interpretation of quantitative structure-function activity relationship data for lactate oxidase
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On the interpretation of quantitative structure-function activity relationship data for lactate oxidase

机译：关于乳酸氧化酶定量构效关系数据的解释

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The native flavin, FMN. has been removed from the L-lactate oxidase of Aerocoew edns, and the apoprotein reconstituted with 1Z FMN derivatives with various substituents at the flavin 6- and 8-positions. Impressive linear relationships are exhibited be- tWeen the sum of the Hammett σ_para and σ_ortho parameters and the redox potentials of the free flavins, and between the redox potentiaIs of the free and enzyme-bound flavins. Rapid reaction kinetics studies of the reconstituted enzymes with the substrates L-lactate and L-mandelate show an increase in the reduction rate constant with increasing redox potential, except that, with lactate, a limiting rate constant of ≈700 s~-1 is obtained with flavins of high potential. Similar breakpoints are found in plots of the rate constants for flavin N5-sulfite adduct formation and for the reac- tion of the reduced enzymes with molecular oxygen. These results are interpreted in terms of a two-step equilibrium preceding the chemical reaction step, in which the second equilibrium step provides an upper limit to the rate with which the particular Substrate or ligand is positioned with the flavin in the correct fashion for the observed chemical reaction to occur. The relation- ship of rate constants for flavin reduction and N5-sulfite adduct formation with flavin redox potential below the observed break- point indicate development of significant negative charge in the transition states of the reactions. In the case of reduction by substrate. the results are consistent either with a hydride transfer mechanism or with the so called "carbanion" mechanism, in which the substrate alpha-proton is abstracted by an enzyme base protected from exchange with solvent. These conclusions are supported by substrate alpha-deuterium isotope effects and by solvent viscosity effects on sulfite binding.
机译：天然黄素，FMN。已从Aerocoew edns的L-乳酸氧化酶中去除了​​脱辅基蛋白，载脂蛋白由在黄素6和8位带有各种取代基的1Z FMN衍生物重构。 Hammettσ_para和σ_ortho参数与游离黄素的氧化还原电位之和以及游离黄素和酶结合黄素的氧化还原电位之间表现出令人印象深刻的线性关系。对重组酶与底物L-乳酸盐和L-扁桃酸盐的快速反应动力学研究表明，还原速率常数随氧化还原电位的增加而增加，不同之处在于，对于乳酸，可获得约700 s〜-1的极限速率常数。具有高潜力的黄素。在黄素N5-亚硫酸盐加合物形成和还原的酶与分子氧的反应的反应速率常数图中可以找到类似的断点。这些结果是根据化学反应步骤之前的两步平衡来解释的，其中第二平衡步骤为特定底物或配体与黄素以观察到的正确方式定位的速率提供了上限。发生化学反应。黄素还原和N5-亚硫酸盐加合物的形成与黄素氧化还原电势低于观察到的断裂点的速率常数之间的关系表明，在反应的过渡态中会产生明显的负电荷。在被底物还原的情况下。结果与氢化物转移机理或所谓的“碳负离子”机理是一致的，在这种机理中，底物α-质子通过被保护不与溶剂交换的酶碱基而被提取。这些结论得到底物α-氘同位素效应和溶剂粘度对亚硫酸盐结合作用的支持。

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