适用于本科教学的新酶促反应动力学实验研究

Research on a novel kinetics experiment of enzyme-catalyzed reaction presented for undergraduate teaching

通过严格的化学反应动力学推导,得到了(-糜蛋白酶催化三甲基乙酸对硝基苯酯水解反应相关的动力学参数的表达式,包括表观米氏常数KM、酶-底物络合物稳定常数K、催化速率常数kcat、以及基元反应步骤速率常k2和k3等。实验在25℃,pH=8.5的三羟基氨基甲烷缓冲液中进行,用分光光度法测定反应的动力学曲线。数据经过非线性拟合和线性拟合,得到相关的动力学参数,该参数与理论预测一致。该实验酶制剂价格低廉,活性稳定,实验方法简单,可作为本科物理化学实验课程中复杂反应动力学测量的一个很好的实例,加深学生对化学反应动力学相关理论和概念的理解和掌握。

The mechanism of a-chymotrypsin-catalyzed hydrolysis of 4-nitrophenyl trimethylacetate includes the following steps：the reversible enzyme-substrate binding and the enzyme acylation and enzyme deacylation.This mechanism is quite different from the conventional Michaelis-Menten mechanism for enzyme-catalyzed reactions.The corresponding kinetic parameters can be expressed precisely on the basis of chemical kinetics theory,such as Michaelis constant KM ,equilibrium constant K,catalytic rate constant kcat ,and the rate constants of the elementary reactions k 2 and k 3 .The reaction is carried out at 25 ＆#176;C in a pH = 8.5 buffer of tris（hydroxymethyl）aminomethane （TRIS）by means of spectrophotometry.The kinetic curves are fitted linearly and non-linearly.The kinetic parameters are in agreement with the theoretical prediction.The enzyme product used in this experiment is cheap and the activity is stable. The experimental method is simple enough to be applied in undergraduate physical chemistry laboratory courses.As an ideal model of complex reaction,the experiment will help the students to enhance the understanding and knowledge of chemical kinetics theories.