探討乙醯膽鹼酯酶在固定化和游離狀態下之酵素動力學

Abstract

在過去的文獻中，關於乙醯膽鹼酯酶(E.C. 3.1.1.7)的研究，大多是探討在游離狀態下的酵素特性變化。但是在生物體中乙醯膽鹼酯脢並不是以游離的狀態存在，而是以鑲嵌的方式固定在細胞膜上，因此酵素的特性可能在不同的生理條件下而有差異。本研究將乙醯膽鹼酯酶固定在二氧化矽晶片上以模擬此酵素固定在細胞膜上的環境。配合實驗室之前開發的量測固定化酵素平台，測量出被固定化的乙醯膽鹼酯酶的酵素動力學參數K*m 和 V*max，探討三維和二維的乙醯膽鹼酯酶在兩種不同狀態下的酵素動力學。本研究利用ＬabVIEW圖形化程式將固定化酵素量測平台過於繁雜的分析過程，開發為自動化計算的人機介面。此程式縮短了繁瑣的計算程序同時在短時間內就能得到固定化酵素的動力學參數K*m 和 V*max。 整合此兩套系統將能夠快速得到研究所需要之固定化酵素動力學參數。乙醯膽鹼酯酶在二維狀態下酵素動力學參數K*m為46□3 □M和V*max為7.2□0.6 □mole/mg/min;而三維狀態下酵素動力學參數Km為79□8 □M和Vmax為159□5 □mole/mg/min。目前治療阿茲海默症的主要方法為，利用藥物抑制乙醯膽鹼酯脢進而減少神經傳遞素，以減緩此症狀的發生，未來本技術將可應用於探討藥物抑制乙醯膽鹼酯酶的活性。

Enzymatic properties of acetylcholinesterase (AChE, E.C. 3.1.1.7) were mainly studied in vitro at it free form in solution. However, the in vitro properties of the enzyme may differ in physiological condition because AChE is immobilized as a membrane anchored protein in the organism. The method of this research was to immobilize AChE on silicon oxide surface in order to imitate the virtual environment of the cell membrane. Based on a measurement platform previously developed by our laboratory, K*m and V*max of immobilized AChE on planar silicon surface were determined. Automatic LabVIEW graphical program were developed to replace previously complicated analyzing processes, meanwhile, generated the kinetic parameters of immobilized enzyme. Kinetic parameters, Km and Vmax, of AChE were 46□3 □M and 7.2□0.6 □mole/mg/min in two dimension (on silicon oxide surface) and 79□8 □M and 159□5 □mole/mg/min in three dimension (in solution), respectively. Nowadays, controlling AChE activity by medicine to reduce acetylcholine is the main solution for treating Alzheimer’s disease. This technique can be used for investigating immobilized AChE activity under the influence of inhibitors that can be applied to the treatment of Alzheimer’s disease.