A hyperthermophilic protein acquires function at the cost of stability
A hyperthermophilic protein acquires function at the cost of stability
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DOI：
10.1021/bi060907v
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发表时间：
2006-10-24
期刊：
影响因子：
2.9
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Mukaiyama, Atsushi;Haruki, Mitsuru;Kanaya, Shigenori
在中等温度下生长的生物体的蛋白质中，活性中心残基通常不会对构象稳定性(活性-稳定性权衡)进行优化。目前尚不清楚活性和稳定性的权衡是否适用于来自高温微生物的蛋白质。由于酶的活性通常在较高的温度下增加，而超嗜热蛋白需要很高的构象稳定性，所以他们可能不会为了自己的活性而牺牲稳定性。本研究试图阐明活性中心残基对超嗜热蛋白质构象稳定性的贡献。因此，我们研究了柯达卡氏热球菌核糖核酸酶HII(TK-RNaseHII)的野生型和活性位点突变蛋白(D7N、E8A、E8Q、D105A和D135A)的热力学稳定性和酶活性。用圆二色谱在220 nm波长处测定了盐酸胍(GdnHCl)诱导的变性，并用差示扫描量热法研究了热诱导变性。在这些蛋白质中，GdnHCl和热诱导的变性都是高度可逆的。除Glu8到Gln外，所有这些活性中心残基的突变都显著降低了酶的活性，但使蛋白质在50℃下的稳定性增加了7.0~11.1kJ·mol(-1)。Glu8到Gln的突变对酶活性影响不大，在50℃下仅增加了2.5kJ·mol(-1)。因此，高温蛋白质的构筑机理等同于常温蛋白质的构筑机理。
Active-site residues are not often optimized for conformational stability (activity-stability trade-offs) in proteins from organisms that grow at moderate temperature. It is unknown if the activity-stability trade-offs can be applied to proteins from hyperthermophiles. Because enzymatic activity usually increases at higher temperature and hyperthermophilic proteins need high conformational stability, they might not sacrifice the stability for their activity. This study attempts to clarify the contribution of active-site residues to the conformational stability of a hyperthermophilic protein. We therefore examined the thermodynamic stability and enzymatic activity of wild-type and active-site mutant proteins (D7N, E8A, E8Q, D105A, and D135A) of ribonuclease HII from Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced denaturation was measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation were highly reversible in these proteins. All the mutations of these active-site residues, except that of Glu8 to Gln, reduced the enzymatic activity dramatically but increased the protein stability by 7.0 to 11.1 kJ mol(-1) at 50 degrees C. The mutation of Glu8 to Gln did not seriously affect the enzymatic activity and increased the stability only by 2.5 kJ mol(-1) at 50 degrees C. These results indicate that hyperthermophilic proteins also exhibit the activity-stability trade-offs. Therefore, the architectural mechanism for hyperthermophilic proteins is equivalent to that for proteins at normal temperature.