A Method for the Kinetic Analysis of Progress Curves Using Horse Serum Cholinesterase As a Model Case

A technique which utilizes the kinetic information present in the progress curve for a reaction has been developed to determine the values of some kinetic parameters and has been used to study the hydrolysis of an artificial substrate, acetyl ester of 3-hydroxyphenyltrimethylammonium bromide by horse serum cholinesterase. In this method, many data points (each defined by a substrate concentration, a product concentration, and a velocity) can be obtained from each progress curve. Data from at least two such curves are then fit to the rate equation for the reaction to determine the values of the kinetic parameters, Km, Vm, KIS, and KII. Such values, determined by this progress curve assay method, proved to be identical with those found by the usual initial velocity assay method. The mechanism of the hydrolysis of the acetyl ester of 3-hydroxyphenyltrimethylammonium bromide by horse serum cholinesterase is ordered Uni-Bi. The reaction was found to have a Km of 0.149 mm and a Vm of 2.21 µmoles nonacetylated phenol from the acetyl ester of 3-hydroxyphenoltrimethylammonium bromide per min per mg of enzyme. One of the two products of the reaction (acetate) was shown to have little or no effect upon the reaction (Ki ∼ 180 mm). The other product was shown to be a linear non-competitive inhibitor of the reaction. The effect of temperature upon the reaction was studied by use of the progress curve assay method. An Ea of 6500 cal per mole and a ΔH* of 5900 cal per mole at 25° was determined for the reaction. Both initial velocity assays and progress curve assays were used to study the effect of pH upon the reaction. The apparent pK of an active group in the enzyme was found to be 5.8 at 25°, and a heat of ionization of 7000 cal per mole was calculated for it. These results are consistent with a histidine being important in the catalytic activity of the enzyme. The effect of ionic strength upon the reaction parameters was investigated with the use of both progress curve and initial velocity assays. Km and KIS were found to increase with increasing ionic strength, while Vm was found to decrease and KII to remain relatively constant with increasing ionic strength. The effects of choline and N,N-dimethylaminoethanol upon the reaction were determined by use of initial velocity assays. Choline was found to be both an alternate product modifier and a dead end inhibitor of the reaction. N,N-Dimethylaminoethanol was found to be an uncompetitive inhibitor of the reaction.