What if I get a negative value for Ki?
If the X intercept of an inhibited velocity series is not between the Y axis and the Km of the control, then data points (1/Vo) should be dropped, beginning with the most dilute [S], until the line shifts to where it is expected to be. Justification for dropping the data is found in the R2 values or in the knowlege that the pipetting accuracy of the smaller volumes may have been poor, especially if a p1000 pipettor was used. Also, we know that the inhibitors are substrate analogs and are thus competitive inhibitors. It is a known shortcoming of the graphical method that the reliable data points are clustered close to the Y axis (in other words, points farther from the origin are less reliable). A negative inhibition constant is only seen when the “inhibitor” is really an activator. The following example illustrates how dropping the farthest data point from the MGP series causes the calculated Ki to make sense.
