混合型尿素酶抑制剂3-羟基-3-(2-羟基-5-氯苯基)丙酰氧肟酸动力学常数测定方法研究

Exploration for Kinetic Constant Determination of 3-( 5-Chloro-2-Hydroxyphenyl )-3-Hydroxypropionylhydroxamic Acid as a Urease Inhibitor with Mixed Inhibition

探讨尿素酶抑制剂3-羟基-3-(2-羟基-5-氯苯基)丙酰氧肟酸动力学常数测定的影响因素,测定其混合型抑制的非竞争属性动力学常数Ki'。通过合理选取尿素浓度来消除其反馈抑制的干扰,并通过采用酶动力学方程直接对反应速率与底物浓度的V-[S]图进行非线性拟合的方法,来测定混合型尿素酶抑制剂的动力学常数Ki'。结果表明,选用适宜的底物浓度可以使基于线性拟合的双倒数曲线更好的相交于一点,虽然能有效的提高求取(混合型抑制的竞争属性动力学常数)Ki的精度,但不能求取Ki';相反,基于酶动力学方程,对相同的实验数据以反应速度V对尿素浓度[S]进行非线性拟合,能有效的同时确定混合型抑制的动力学常数Ki和Ki'。

Influence factors were explored for the kinetic constant determination of 3-（5-Chloro-2-hydroxyphenyl）-3-hydroxypropio- nylhydroxamic acid （ PPHA ） , a urease inhibitor identified in our group, which inhibited Helicobacter pylori urease with a mixed inhibitory kinetics. Methods were also established to determine the parameter of the non-competitive aspect （Ki＇） of PPHA. To determine the kinetic constant of non-competitive aspect （ Ki＇ ） of the mixed type urease inhibitor PPHA, urea concentration must be carefully selected to eliminate the interference of substrate inhibition ,followed by a nonlinear fitting of experimental data to its integrated Michaelis-Menten equation. Fitting results revealed that the Lineweaver-Burk plots based on a standard linear regression analysis merged well when the urea concentrations were set in an appropriate range, giving the parameter of the competitive aspect （ Ki ） of PPHA with a large improvement but a failure of K＇i. To the contrary, on the basis of same data, nonlinear fitting of velocity against the urea concentration provided both Ki and K＇i in a simple way.