Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence
Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence
复制标题
单氨基酸突变改变短小芽孢杆菌碱性蛋白酶的热稳定性：热力学和温度依赖性
DOI：
10.1093/abbs/gmu120
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发表时间：
2015-02-01
影响因子：
3.7
通讯作者：
Feng, Hong
中科院分区：
文献类型：
--
作者：
Huang, Rong;Yang, Qingjun;Feng, Hong
短小芽孢杆菌BA06脱毛碱性蛋白酶(DHAP)具有催化效率高、热稳定性好等优点，在皮革加工中具有广阔的应用前景。为了深入了解其催化机理，根据同源模建和多序列比对的方法，对两个单一氨基酸取代的突变体进行了热变性热力学和底物水解度的温度依赖性表征。结果表明，在较宽的温度范围内，V149I和R249E的催化效率(k(CAT)/K-m)都有系统的提高，这主要是由于V149I的k(1)(底物扩散)和k(2)(酰化)以及R249E的k(2)和k(3)(脱酰)的增加。与野生型DHAP相比，V149I的热稳定性提高，R249E的热稳定性降低。热力学分析表明，热变性的活化能(ΔG(A)度)可能控制热稳定性。对于V149I，增量G(A)度值增大，而对于R249E，增量G(A)度值减小。基于这些数据和结构模拟，我们认为用Ile取代Val149可能会扰乱底物结合口袋的局部柔性，从而提高与底物的结合亲和力。相反，用Glu取代Arg249会导致与酶的C末端的相互作用中断，从而导致热稳定性降低。本研究表明，在细菌碱性蛋白酶中，活性中心或底物结合口袋中的氨基酸残基可能会干扰催化过程，可作为蛋白质工程的靶标。
Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (k(cat)/K-m) in a wide range of temperatures, mainly due to an increase of k(1) (substrate diffusion) and k(2) (acylation) for V149I and of k(2) and k(3) (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (Delta G(a)degrees) of activation for thermal denaturation may govern the thermostability. The value of Delta G(a)degrees is increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.