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随着基因技术的发展,人们几乎已经可以任意替换蛋白质序列中的特定残基。由于理论上对蛋白质性能的预测工作还远远跟不上实验的发展,只能花费大量的人力、物力利用实验方法筛选突变体。这就迫切需要建立一套能够正确预测蛋白质性质的方法。静电作用在酶促反应中起到了重要作用。基于表面带电残基对酶活性部位残基pK_α的影响,可利用替换表面残基的方法改变酶促反应随pH的变化趋势,是蛋白质工程研究中的一种有效方法。由于表面极性残基处于高介电常数的溶剂(水)与低介电常数的蛋白质的界面上,对此问题尚未有圆满的理论方法进行解决。我们利用Kirkwood理论定量地表述了表面荷电残基的替换对酶活性部位残基pK_α的影响,为研究酶突变体的性质,如突变体的电化学性质、酶催化反应随pH变化趋势的改变,酶专一性的改变等提供了一种简便的方法。
With the development of genetic technology, it is almost impossible to replace any specific residue in a protein sequence. As the theoretical prediction of protein performance is far behind experimental development, it can only spend a lot of manpower and material resources to screen mutants using experimental methods. This urgently needs to establish a set of methods that can correctly predict the nature of the protein. Electrostatic interaction plays an important role in the enzymatic reaction. Based on the effect of surface charged residues on the pK_α of the enzyme active site, the method of replacing the surface residues can be used to change the trend of the enzymatic reaction with pH, which is an effective method in protein engineering. Due to the fact that surface polar residues are located at the interface between a high dielectric constant solvent (water) and a low dielectric constant protein, no satisfactory theoretical solution to this problem has yet been reached. We used Kirkwood’s theory to quantitatively describe the influence of the substitution of surface charged residues on the pK_α of the active site of the enzyme. In order to study the properties of enzyme mutants, such as the electrochemical properties of the mutants and the change of the enzymatic reaction as the pH changes , Enzyme-specific changes provide a convenient way.