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

Abstract:

Objective：To estimate Michaelis-Menten constant（Km） and maximal reaction rate（Vm） of an enzyme through the integra－tion of kinetic analysis of reaction curve for the ratio of maximal reaction rate（Vm） to Michaelis-Menten Constant（Km） as Vm/Km with the initial rate（Vi）. Methods：Bacillus fastidiosus uricase（BFU） and its mutants（A1R，A1R/V144A），calf intestinal alkaline phos－phatase（CIAP） and Escherichia coli alkaline phosphatase mutant R168K served as the models. Reaction curve of uricase was recorded as absorbance at 293 nm with uric acid as the substrate while that of alkaline phosphatase was monitored by absorbance at 450 nm with 4-nitro-1-naphthylphosphate（4NNPP） as the substrate. Vm /Km was estimated by nonlinear fitting of the integrated Michaelis-Menten rate equation with the predictor variable of reaction time to a reaction curve at a substrate concentration smaller than 10% of Km，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 sub－strate concentrations lower than Km for the same enzyme quantity is suitable for the estimation of Vm and Km.