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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Biocatalyst Activity In Nonaqueous Environments Correlates With Centisecond-range Protein Motions
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Biocatalyst Activity In Nonaqueous Environments Correlates With Centisecond-range Protein Motions

机译：非水环境中的生物催化剂活性与厘米范围的蛋白质运动相关。

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摘要

Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, ~1H NMR relaxation, and ~(19)F NMR spectroscopy experiments were performed on subtilisin Carls-berg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive ΔΔS~+, for the more active biocatalyst preparations in organic solvents. In contrast, ΔΔH~+ was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and ~1H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal magnetization exchange, revealed that the enzyme's turnover number (k_(cat)) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the millisecond regime, and uncorrelated with protein motions on the piconanosec-ond timescale. In addition, ~(19)F chemical shift measurements and hyperf ine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphi-nyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity.
机译：最近的研究探索了酶催化与水相中蛋白质动力学之间关系的研究，已经得出证据表明动力学和酶活性密切相关。鉴于蛋白质动力学在有机溶剂中显着减弱，并且蛋白质根据特定的溶剂环境表现出广泛的运动，因此非水环境为检验蛋白质动力学在酶活性中的作用提供了独特的机会。对枯草杆菌蛋白酶Carls-berg与几种无机盐共冻干并悬浮在有机溶剂中进行了可变温度动力学测量，X带电子自旋共振光谱，〜1H NMR弛豫和〜（19）F NMR光谱实验。结果表明，对于有机溶剂中活性更高的生物催化剂制剂，盐活化会诱导更高程度的过渡态柔性，这由更正的ΔΔS+反映出来。相反，无论盐类型或盐含量如何，ΔΔH〜+都可以忽略不计。电子自旋共振谱和〜1 H NMR弛豫测量，包括自旋-晶格弛豫，旋转框架中的自旋-晶格弛豫和纵向磁化交换，表明酶的周转数（k_（cat））与蛋白的运动密切相关。毫秒级时间制，与毫秒级蛋白质运动微弱相关，与皮秒级时标上的蛋白质运动不相关。此外，分别被4-氟苯磺酰氟和4-乙氧基氟膦基-N-氧基-TEMPO抑制的生物催化剂配方的〜（19）F化学位移测量和超精细张量测量表明，酶活化仅受活性变化的微弱影响位极性。

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