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

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。

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

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.