低于米氏常数底物浓度下酶动力学参数的测定

An integration strategy for the estimation of kinetic parameters of an enzyme at substrate concentrations lower than Michaelis-Menten constant

目的:联用设定底物浓度下初速度(initial velocity,V_i)和过程分析法所得酶最大反应速度(maximal reaction rate,V_m)与米氏常数(Michaelis-Menten constant,K_m)比值(V_m/K_m),据米氏方程推算酶的K_m和V_m。方法:以苛求芽孢杆菌尿酸酶(Bacillus fastidious uricase,BFU)及其突变体(A1R,A1R/V144A)、牛小肠碱性磷酸酶(calf intestinal alkaline phosphatase,CIAP)及大肠杆菌碱性磷酸酶突变体R168K为模型。用A_(293 nm)记录尿酸酶反应曲线、A_(450 nm)记录碱性磷酸酶水解4-硝基-1-萘基磷酸酯(4-nitro-1-naphthyl phosphate,4NNPP)反应曲线;以反应时间为自变量、用积分速度方程拟合不超过10%K_m初始底物浓度下酶反应曲线测定V_m/K_m,分析设定底物浓度下初速度反应数据确定初速度V_i;据相同酶量V_i和V_m/K_m按米氏方程计算K_m;据所得V_m/K_m和K_m推算V_m。结果:以E-H(Eadie-Hofstee)法分析50%~200%K_m底物浓度下V_i所得K_m均值为其参考值;联用策略测定V_i所设定的底物浓度越高,则所得K_m相对偏差越小;测定V_i所设定的底物浓度从35%K_m逐渐升高到120%K_m,所得K_m趋于稳定且相对偏差不超过20%。据此联用策略发现,苛求芽孢杆菌尿酸酶在生理pH下活性降低的原因不是其K_m明显升高,而是其V_m下降。结论:当底物溶解度低于K_m时,这种联用策略更适合测定酶的K_m和V_m。

Objective :To estimate Michaelis-Menteh constant(Kin)and maximal reaction rate (Vm)of an enzyme through the integration of kinetic analysis of reaction curve for the ratio of maximal reaction rate(Vm)to Michaelis-Menten Constant(Kin)as Vm/Km with the initial rate (Vi).Methods:Bacillus fastidiosus uricose (BFU)and its mutants (A1R,A1R/V144A),calf intestinal alkaline phosphatase(CIAP)and Escherichia coli alkaline phosphatase mutant R168K served as the models.Reaction curve of uricase was recorded as absorbance .at 293nm with uric acid as the substrate while that of alkaline phosphatase was monitored by absorbanee at 450nm with 4-nitro-l-naphthylphosphate (4NNPP)as the substrate.Vm/Km Was estimated by norrtinear fitting of the integrated Miehaelis- Menten rate equation with the predictor variable of reaction time to a reaction curve at a substrate concentration smaller than 10% of Kin,and Vi was estimated from data for initial rate reaction at a preset substrate concentration.In the integration strategy,Km was derived from the ratio of Vi to Vm/Km for the same enzyme quantity and Vm was .thus derived from known Vm /Km and Km.Results: Reference values of Km were estimated with substrate concentrations ranging from about 50% Km to about two-fold of Km by Eadie- Hofstee transformation of data.In the integration strategy,the use of a higher preset substrate concentration to estimate Vi gave Km closer to the reference value;when the substrate concentrations preset to estimate Vi were varied from 35%to 120%of Km,the derived Km was consistent with the reference value.By this integration strategy under optimized conditions,it was found that the decrease of the activities of BFU and its mutants at physiological pH was caused by the decrease of their Vm rather than the increase of their Km.Conclusion:The integration of Vm /Km estimated through kinetic analysis of reaction,curve with the initial rates at preset substrate concentrations lower than Km for the same enzyme quantity is suitable for the estimation of Vm and Km.