分光光度法研究EDTA对小牛肠碱性磷酸酶的不可逆抑制动力学

Kinetics of Inactivation of Calf Intestine Alkaline Phosphatase by EDTA with Absorption Spectrum Method

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。

Calf intestinal alkaline phosphatase (EC. 3.1.3.1) is a dimeric metalloenzyme composed of two identical subunits, the each active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied for a study on the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenylphosphate (PNPP) and inactivator EDTA suggested a competitive complexing mechanism for inactivation by EDTA, and the process of inactivation composed of the rapid initial formation of an enzyme-EDTA complex, in which the conformation of enzyme has been changed, and then zinc ions are finally removed from the enzyme.