【摘要】 运用动力学方法对巨大芽孢杆菌 (Bacillusmegaterium)来源的重组青霉素G酰化酶及其突变体的 pH依赖性催化反应机制进行了研究 ,从logVm和log(Vm/Km)与 pH关系曲线计算得到野生型青霉素G酰化酶参与催化反应的离子化基团的 pK1和 pK2 分别为 5 .5 0～ 5 .87和 10 .73。研究结果表明 ,两种突变体的 pK1和 pK2 值与野生型很接近 ,突变体A和B的pK1分别为 5 .6 4～ 5 .86和 5 .6 9～ 6 .96 ,pK2 分别为 10 .6 1和 10 .4 8。与此同时还考察了不同反应温度时野生型青霉素G酰化酶的 pK1和pK2 值 ,从 pK1和 pK2 与温度的关系曲线计算可得离子化基团的解离热焓ΔH分别为 4 4 .38～ 5 9.0 3kJ/mol和 14 7.37kJ/mol。根据上述实验结果 ,推测两个参与催化反应的离子化基团可能是位于活性中心附近的组氨酸和赖氨酸。更多还原

【Abstract】 The pH-dependence in the catalytic reaction of recombinant penicillin G acylase and its mutants fromB.megaterium has been studied by using kinetic methods. pK 1 and pK 2 of the residues of the wild type penicillin G acylase, involved in the catalyzed reaction, were 5.50—5.87 and 10.73, respectively, from the curves of logV m and log(V m/K m) versus pH. Results showed that the pK 1 and pK 2 values of these residues of the mutants were similar to that of the wild type. pK 1 of 5.64—5.86 for mutant A and 5.69—6.96 for mutant B were obtained, while pK 2 was 10.61 and 10.48 for mutant A and B, respectively. At the same time, pK values at different temperatures were investigated. The ionization enthalpies(ΔH) were 44.38—59.03 kJ/mol and 147.37 kJ/mol, respectively, from the curve of pK versus temperature. It was presumed according to the results mentioned above that the ionizing residues, involved in the reaction, were histidine and lysine that are localized around the active site.更多还原