Title:An Additional Method for Analyzing the Reversible Inhibition of an Enzyme Using Acid Phosphatase as a Model
VOLUME: 11 ISSUE: 2
Author(s):Jordan M. Baumhardt, Benjamin M. Dorsey, Craig C. McLauchlan and Marjorie A. Jones
Affiliation:Department of Chemistry, Illinois State University Normal, IL 61790-4160, USA.
Keywords:Acid phosphatase, enzyme kinetics, inhibition mathematical model.
Abstract:Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a
novel method for analyzing reversible inhibition was carried out. Our alternative approach involves
plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration
to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The
concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants
can be derived from this plot: Kmax, Kmin, and Kinflect. Kmax and Kmin represent the substrate to inhibitor
concentration ratio for complete inhibition and minimal inhibition, respectively. Kinflect represents
the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to
the inhibitory complex. These constants can be interpolated from the graph or calculated using the first
and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis
values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this
means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with
substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with
concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot
appears useful for known competitive and non-competitive inhibitors and may have general applicability.
