Department of Biochemistry;
Chongqing University of Medical Sciences;
Chongqing 400016;
China;
Department of Pharmacology;
Chongqing University of Medical Sciences;
Chongqing 400016;
China;
Department of Biochemistry;
Chongqing University of Medical Sciences;
Chongqing 400016;
Chinabjective: To investigate the reliability for fast estimation of Michaelis-Menten constant ( Km ) with calibrated specific activity at only two medium concentrations of substrate by both simulation and experimentation with aryles-terase (ArE)as model. Methods: Initial rates were simulated by randomly inserting uniform absolute error;
and the experimental initial rates of ArE were determined by measuring the increaser of product absorbance. Calibrated specific activities at two substrate concentrations were obtained by regression analysis;
and Km was calculated according to Michaelis-Menten equation. Results: By simulation with calibrated specific activities at two medium substrate concentrations;
Km could be calculated according to Michaelis-Menten equation with reasonable precision and accuracy. By experimentation with substrates of 2-naphthyl acetate;
phenyl acetate;
and p-nitrophenyl acetate;
there were no differences between the mean and SD of Km of ArE for either substrate by this linear kinetic method and the Lineweaver-Burk plot. Conclusion: This linear kinetic method was reliable for fast estimation of the Km of some specified enzyme on its substrate of lower solubility or lower sensitivity for quantification by common methods.;