Direct determination of kinetic parameters for the hydrolysis of a poor substrate by human plasma

A method for characterizing the kinetic parameters of poor substrates is proposed. The hydrolysis of succinyldicholine by human wild type butyrylcholinesterase from whole plasma was chosen as a model reaction. Since the poor substrate succinyldicholine shows an inhibitory effect on butyrylcholinesterase hydrolysis of the enzyme's optimal substrate butyrylthiocholine, its inhibition constant was determined in the first of three steps, using Ellman's colorimetric detection method. In the second step, this inhibition constant is used to calculate the remaining succinyldicholine concentrations after incubating the enzyme at known initial concentration for various time periods. Finally, the catalytic constant is determined by numerically integrated Michaelis-Menten equation using data for the time course of the residual succinyldicholine concentration. This assumption was then confirmed by simultaneously analyzing all three data sets using the "ENZO" fitting tool. Additionally, the results are compared with those collected by another colorimetric method utilizing bromthymol blue to follow a small change in pH during the course of substrate hydrolysis. In this case the hydrolysis of succinyldicholine to succinylmonocholine and subsequently to succinic acid can only be followed by highly concentrated purified wild-type human butyrylcholinesterase. Close values of parameters, determined by two different methods, suggest the approach for revealing enzymes responsible for the metabolization of other xenobiotics, too.