It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity–flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (βα)₈-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop β1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

中文翻译：

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（βα ）_8-桶酶吲哚-3-甘油磷酸合酶的催化作用与活性位点环动力学的关系

重要的是要了解酶的催化活性如何与其构象柔韧性相关。我们以Sulfolobus solfataricus（ssIGPS）的吲哚-3-甘油磷酸合酶为例研究了这种活动-灵活性相关性，该酶催化色氨酸的生物合成中的第五步。ssIGPS是酶的热稳定代表，具有常见的催化通用（βα）₈-桶折。通过在25°C下的瞬态动力学分析了具有更高催化转化率的ssIGPS的四个变体，同样在25°C和60°C下也分析了野生型ssIGPS。具有最小三步模型的全局拟合为底物结合，化学转化和产品释放提供了单独的速率常数。结果表明，在两种情况下，即激活突变的应用和温度的升高，相对于化学转化步骤而言，产物释放速率的加速都提供了催化周转数的净增加。在25°C和60°C下对溶剂粘度影响的测量结果证实了速率确定步骤中温度的这种变化，这与ssIGPS的Arrhenius图在约40°C下的扭折相一致。以Arrhenius图的形式绘制附着在活性位点环β1α1上的顺磁性电子自旋标记的旋转扩散率时，在相同温度下观察到扭结。这些发现以及分子动力学模拟表明，不同程度的环迁移率与ssIGPS催化机理中的不同限速步骤相关。