The graphical analysis of steady-state kinetic data according to Eadie-Augustinsson-Hofstee (EAH plot) is illustrated for multisubstrate systems and compared with the double reciprocal plot (Lineweaver-Burk plot) commonly used. It is emphasized that the choice of graphical representation may be of crucial importance when a discrimination between alternative rate laws has to be made. A major advantage of the EAH plot is the high sensitivity to deviations from linearity at low substrate concentrations.
It is well established that rigorous evaluation of enzyme steady-state kinetic data requires statistical treatment (l-3). Statistical criteria can then be used to distinguish between different plausible mechanisms of the enzymatic reaction investigated (4,5). Preferably, the kinetic parameters, such as K, and V, are estimated by computer fitting of the assumed rate equation to the experimental data. Simple hyperbolic rate equations can by suitable transformations be analyzed by linear regression. In the latter case, minimum bias in the parameter estimation is obtained by regarding S as a function of S/v (6). However, quite apart from the parameter estimation, graphical analysis is always useful in the treatment of the more fundamental question, viz., the discrimination between alternative rate equations. This aspect makes the choice of plotting method for analysis of the data a matter of major importance, because features appearing in a particular type of plot may not be readily recognized in another. For the purpose of discrimination a linear representation of the rate law considered is normally preferred, because deviations from linearity can often be discerned by inspection of plots even in cases where no statistical significance can be obtained for the curvature. In such cases proper experiment design (5,6) may yield new data and a clear-cut discrimination.
For a one-substrate reaction the v versus v/S plot (ascribed to Eadie 12