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应用了邹氏不可逆抑制动力学的方法,在变性剂存在的条件下,连续监测酶催化的底物反应过程,测定了氨基酰化酶在不同浓度脲溶液中失活的速度常数,并考察了底物的存在对酶失活的保护作用。同时,还比较了氨基酰化酶在不同浓度脲溶液中变性时失活和构象变化的速度。1mol/L,2mol/L脲变性时失活速度常数为为1.62×10~(-2)S~(-1)和2.05×10~(-2)S~(-1),但是酶分子的整体构象尚未发生明显变化。3mol/L脲变性时,失活速度常数为2.56×10~(-2)S~(-1),而变性速度常数为3.72×10~(-3)S~(-1)。失活速度比构象变化速度快大约一个数量级。在4mol/L,5mol/L,6mol/L脲溶液中变性时,酶分子快速失活,而其构象变化速度常数分别为5.27×10~(-3)S~(-1)。5.47×10~(-3)S~(-1),5.56×10~(-3)S~(-1)。可见,在相同浓度的脲溶液中,氨基酰化酶的失活速度明显快于酶分子整体构象变化的速度。上述结果表明,含有辅基金属离子Zn~(2+)的氨基酰化酶的活性部位较酶分子的整体结构也具有一定的柔性。
The method of irreversible inhibition kinetics of Zou was applied. The enzyme-catalyzed substrate reaction was continuously monitored in the presence of a denaturant and the rate constants of inactivation of the aminoacylase in various concentrations of urea solution were determined. The presence of substrate protects against enzyme inactivation. At the same time, the rate of inactivation and conformational change of aminoacylase in denaturation at different concentrations of urea solution was also compared. The deactivation rate constants of 1 mol / L, 2 mol / L urea denaturation were 1.62 × 10 -2 S -1 and 2.05 × 10 -2 S -1, The overall conformation has not changed significantly. The deactivation rate constant was 2.56 × 10 -2 S -1 and the denaturation rate constant was 3.72 × 10 -3 S -1 when 3 mol / L urea was denatured. The deactivation rate is about an order of magnitude faster than the conformational change. When denatured in 4mol / L, 5mol / L, 6mol / L urea solution, the enzyme molecules were rapidly inactivated and their conformational constants were 5.27 × 10 -3 S -1. 5.47 × 10 ~ (-3) S ~ (-1), 5.56 × 10 ~ (-3) S ~ (-1). Visible, in the same concentration of urea solution, aminoacylase inactivation rate was significantly faster than the overall rate of enzyme molecule conformational changes. The above results show that the active site of the aminoacylase containing Zn 2+ is more flexible than the overall structure of the enzyme molecule.