Specificity of esterases. I. Identification of two pancreatic aliesterases.

A plot of some linear relationship between reaction rate (v) and substrate concentration (S) is commonly used to estimate the constant,s of an enzyme system that follows the Michaelis-Menten theory of enzyme action. In cases in which there is certainty that only one enzyme is involved, it suffices to apply such a plot over a relatively short range of substrate concentrations. This certainty does not often exist, however. Short range plots then have little meaning, even if reasonable linearity is obtained, since they represent part of a curve which as a whole may have a curvature. On the other hand, a non-linear curve, being the result of the presence of two or more enzymes acting simultaneously on the same substrate while the constants are different, can be used for identification of the enzymes. -4 condition for such a procedure is that a plot is used in which equal emphasis is laid upon all experimental data. Unfortunately, the plots most commonly used (l/S versus l/v and S versus S/u) do not satisfy this condition and as a result do not represent a simple addition of the individual curves (1). The only plot of the Michaelis-Menten equation where the individual enzyme systems contribute to the resulting curve in direct proportion to the magnitude of their constants is a plot of v against v/S (l), based on the equation V,,, = v + vKM/S. A further condition is that the method of activity determination allows the measurement of initial reaction rates over a wide range of substrate concentrations. In the present investigation, the esterolytic activity of a pancreatic lipase preparation (steapsin) with respect to valerylsalicylic acid was determined over a loo-fold range of the substrate concentration. The slope of the curve obtained from a plot of v against v/S increased about ten times from the lowest to the highest substrate concentration used. This indicated the presence of two or more enzymes with different KM values. After isolating one of the enzymes, it could be shown that the original curve was a result of simultaneous action of essentially two esterases of which the constants V,,, and KM were ent,irely different. It was shown