截流荧光法研究米曲霉氨基酰化酶的快速胍变性动力学

 用荧光光谱法、截流荧光法和酶活力测定法研究了在盐酸胍溶液中米曲霉氨基酰化酶变性动力学。我们发现在4.8mol/L盐酸胍溶液作用下(0.05mol/L磷酸缓冲溶液,pH7.4,25℃),氨基酰化酶二聚体解离成单亚基过程是一个十分快速的过程,反应速率常数k为3361l/s,即约需3ms时间完成;而单亚基分子的构象变化需要约20min方能到达平衡态,这是一个逐渐变化的缓慢过程。酶分子在胍作用下的失活现象同酶分子的结构变化紧密相关,在胍浓度大于4mol/L时酶完全失活。在高浓度盐酸胍下酶失活主要是因为酶二聚体迅速解离成单亚基的过程和单亚基构象逐渐变化的缓慢过程。双亚基解离常数大小标志着酶分子亚基间作用力的强弱。

A Kinetic Study of Rapid Denaturation of Aminoacylase From Aspergillus Oryzae in Guanidine by Stopped-Flow Fluorescence

The Kinetics of denaturation of aminoacylase from A.Oryzae in guanidine hydrochloride solution has been studied by fluorescence, Stopped-flow fluorescence and enzme activity measurements. It was found that in 4.mol/L guanidine HCI (0.05mol/L phesphate buffer, pH7.4, 25℃), the process of dimer dissociation into monomer is a fast phase,and the velocity constant k is 336 sec-1, inwhich the dissociation takes only 3 milliseconds to complete. However, the conformation change of the monomer needs about 20 minutes to reach the stable state and this is a relatively slow phase.There is a better relationship between the inactiviation and conformation change of the aminoacylase in guanidine solution of more than 4 mol/L in which the aminoa-cylase will be inactiated completely.The inactivation of aminoacylase in guanidine solution was caused by rapid dissociation of dimer into monomer which was then slowly changed into a stable conformation. The velocity constant k of dimer dissociation reflects the strength of interaction between the enzyme subunits.