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运用邹氏酶活性不可逆改变动力学 ,在不同酶底物浓度及不同抑制剂浓度下 ,用分光光度法监测酶底物水解产物浓度随时间变化的过程 ,研究了抑制剂EDTA对小牛肠碱性磷酸酶活性的不可逆抑制作用。结果表明 :EDTA对小牛肠碱性磷酸酶抑制反应机理为 :EDTA与小牛肠碱性磷酸酶发生络合作用 ,形成中间态的酶 EDTA络合物 ,此络合物的形成 ,导致该酶活性中心微环境构象发生变化 ,使酶的催化活性丧失 ,随后EDTA将酶中金属离子拉出 ,使酶发生不可逆失活。实验测定了 3 7℃下EDTA对小牛肠碱性磷酸酶不可逆抑制作用的微观速率常数ki 为 0 0 5 2 9s-1及EDTA与酶结合的平衡常数KI 为 4 0 0mmol·L-1。
Using the irreversible kinetics of ZO activity, the enzyme substrate hydrolyzate concentration was monitored by spectrophotometry at different enzyme substrate concentrations and different inhibitor concentrations. The effect of inhibitor EDTA on calf intestinal Irreversible inhibition of phosphatase activity. The results showed that EDTA inhibited calf intestinal alkaline phosphatase reaction mechanism: EDTA and calf intestinal alkaline phosphatase complexation, the formation of the intermediate enzyme EDTA complex, the formation of the complex, leading to the Enzyme activity changes in the microenvironment of the center of the environment, so that the loss of catalytic activity of the enzyme, followed by EDTA to pull out the metal ions in the enzyme, the enzyme irreversible inactivation. The micro-rate constant ki of EDTA at 37 ℃ under the irreversible inhibition of calf intestinal alkaline phosphatase was 0 0 5 2 9s-1 and the equilibrium constant of EDTA-enzyme binding KI was 400 mmol·L -1.