甲基对硫磷水解酶(methyl parathion hydrolase,MPH)能催化甲基对硫磷等有机磷化合物中P-O、P-F、P-CN及P-S键的水解。该研究考查了K+对MPH催化活性的影响,并分析了K+调控酶蛋白活性的作用位点。酶活分析显示,Pseudomonas sp. WBC-3 MPH催化活性随K+浓度增加而迅速提高,MPH 在3 mol/L K+溶液中的催化活性较无K+条件下提高13.39倍。基于MPH进行分子动力学分析,建立了MPH氨基酸残基临近区域K+分布概率的预测方法。预测结果表明,1 mol/L K+溶液中K+倾向分布于MPH的Asp、Glu、Gln及Asn残基附近,并降低了A85-T95与V323-N329区域的柔性。将A85-T95与V323-N329柔性变化最大的E94与N329分别突变为Ala,使MPH相对活性(1 mol/L K+溶液/0 mol/LK+溶液)分别较野生酶降低26%与33%;两者同时突变为Ala,其催化活性降低53%。上述结果表明,K+通过与E94、N329相互作用来调控MPH催化,为其催化活性的分子改造提供了靶点。
Methyl parathion hydrolase (MPH) can catalyze the hydrolysis of P-O, P-F, P-CN and P-S bonds in organophosphorus compounds such as methyl parathion. In this study, the effect of K+ on the catalytic activity of MPH was investigated, and the residues interacting with K+ on the activity of MPH were also analyzed. Enzymatic activity analysis showed that the activity of MPH increased rapidly with the increase in ion concentration of potassium. The catalytic activity of MPH in 3 mol/L K+ solution was 13.39 times higher than that in the absence of K+. Based on the molecular dynamic analysis of MPH, the prediction method of K+ distribution in the vicinity of amino acid residues of MPH was established. The predicted results showed that K+ tends to distribute near Asp, Glu, Gln and Asn residues of MPH, and reduced the flexibility of A85-T95 and V323-N329 regions. The most flexible residues E94 and N329 in these two regions were mutated into Ala, which reduced the relative activity of MPH (1 mol/L K+/ 0 mol/L K+) by 26% and 33% respectively and mutating both the residues into Ala reduced the relative activity by 53%. These results indicated that K+ regulated MPH reaction by interacting with E94 and N329, which provides target residues for molecular modification to enhance MPH activity.
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