of the criteria here as they are covered extensively in the above reference. In
addition, the reader is encouraged to consult the reference Silverman (1998) for
the proper procedures to follow in carrying out studies of mechanism-based
enzyme inactivation.
As a first step in determining the kinetics of mechanism-based enzyme
inactivation, the time-dependence of the reaction is typically studied. In
these experiments, the effects of preincubation (primary incubation) with
increasing concentrations of the inactivator over various time intervals is
studied for their effects on catalytic activity of the enzyme toward the reporter
substrate. This typically results in a plot similar to that represented in
Figure 4.11.
From this plot the half-lives (rate constants) for the inactivation by each
concentration of inactivator can be calculated from the slopes of the individual
lines. These half-life values are then plotted along they-axis versus 1/[I] plotted
along thex-axis. This plot is also known as a Kitz–Wilson plot (Fig. 4.12). In
the case of a saturation reaction, (i.e., at infinite inactivator concentration there
is a finite half-life) the point where the plotted line intersects they-axis is equal
to 0.693/kinact, wherekinactis the rate of inactivation and represents a complex
mixture ofk 2 ,k 3 andk 4 (see Scheme 4.5). The dissociation constant for the
enzyme-mechanism-based inactivator complex (KI) can also be estimated from
this plot as thex-intercept of the line represents1/KI(Fig. 4.12).
Acknowledgment
Portions of this work were funded by a grant from the National Institutes of
Health (#GM 063215).
FIGURE 4.12 Kitz–Wilson plot of the half-lives (rate constants) for mechanism-based
inactivation at each inactivator concentration.
110 ENZYME KINETICS