The Determination of Enzyme Dissociation Constants

Abstract

Graphical methods involving constant slopes and straight line extrapolations have been developed for testing and interpreting kinetic data, and for determining dissociation constants of enzyme-substrate and enzyme-inhibitor compounds and other related constants when the data are found to be consistent with an assigned mechanism. Representative analyses are given for invertase, raffinase, amylase, citric dehydrogenase, catalase, oxygenase, esterase and lipase, involving substrate activation, substrate inhibition, general competitive and non-competitive inhibition, steady states and reactions of various orders. A plot of the reciprocal of the observed velocity v against the reciprocal of the substrate concentration (S) yields in the simplest case (e. g., invertase, amylase) a straight line whose slope and ordinate intercept yield Ks (Michaelis dissociation constant) and Vmax (theoretical maximum velocity). In the presence of competitive inhibitors the slope is increased but the intercept is unchanged. With non-competitive inhibitors the intercept also is raised. When the active intermediate contains n molecules of S a straight line is obtained upon plotting the reciprocal of (S)n, instead of the reciprocal of (S), which would yield a curve concave upward. A steady-state occurring before the formation of the active intermediate, to whose concentration v is proportional, will yield a curve with two limiting slopes. Curves of v plotted directly against (S), passing through an optimum and approaching a zero value, indicate the existence of an additional inactive intermediate (catalase), whereas approaching a constant value indicates two or more active intermediates (l-ethyl mandelic esterase). The constants involved in the three latter cases are determined from limiting slopes and intercepts of various plots. The various methods described are applicable to general chemical catalysis, homogeneous or heterogeneous, and possess many advantages of usefulness and convenience over less extensively developed methods employed heretofore. They are capable of eliminating certain postulated mechanisms, and indicating what mechanism or mechanisms may be involved, though not necessarily proving what mechanism is involved, in a given case. © 1934, American Chemical Society. All rights reserved.