The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature. In this paper we focus on three points(1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature.

Results can be summarized as follows(a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0. We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0. Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14. 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0.

译文

研究了硫酸亚砜中吲哚-3-甘油磷酸合酶的稳定性和活性随pH和温度的变化。在本文中,我们重点关注三点 (1) 蛋白质在高温下不可逆变性的长期稳定性; (2) 蛋白质在可逆温度驱动的去折叠的短期稳定性; (3) 其活性对温度的依赖性。
结果可以总结如下 (a) 在不同的ph值 (6.5,8.0和9.5) 来自80 ℃ 下的长期稳定性实验; (b) 短期稳定性实验揭示了在两个不同pH范围 (6.5-8.0,8.5-9.5) 下的不同行为,表明在碱性下的熔融温度高于在中性pH下; (c) 在pH 7.0和9.0下研究了活性对温度的依赖性,在pH 9.0下的kcat值的Arrhenius图中观察到不连续性。我们还研究了氯化胍在20 ℃ 下在pH 7.0或pH 9.0下的稳定性,并且我们提供的数据表明,展开机制在pH 7.0下接近两态模型,在pH 9.0下接近更复杂的机制。在pH 9.0下通过荧光测量获得的平衡展开转变的令人满意的拟合需要一个模型,该模型除了天然和展开形式之外还涉及稳定的中间体。在20 ℃ 下,折叠构象比未折叠构象在pH 7.0下稳定 (14. 7 +/-1.2) kJ/mol,在pH 9.0下稳定 (25.5 +/-1.8) kJ/mol。

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