than theKm. In this case, the [S] term drops out of the denominator of Eq. 4.1
and the equation reduces to
nVm½S
Kmð 4 : 2 Þsuch that velocity becomes proportional to [S]. It is also well established that
the velocity (or rate of metabolism) of the reaction is equal to the product of
intrinsic clearance and substrate concentration (Eq. 4.3).
v¼CLint½Sð 4 : 3 ÞThis intrinsic clearance (CLint) is analogous to thein vivointrinsic clearance
in that it is the ability of the enzyme to clear (metabolize) drug in the absence of
blood flow or protein binding restrictions. Equation 4.2 is analogous to
Equation 4.3 and thus equality is frequently assumed such that
CLint¼
Vmax
Km¼
v
½Sð 4 : 4 ÞFor a complete discussion of these assumptions, see the excellent report of
Houston (1994).
The ability to estimate CLintfromin vitrodata thus becomes important in
makingin vitro–in vivocorrelations to estimate the ‘‘first dose in man’’ andin
vivodrug disposition. The more accurate the prediction fromin vitrodata, the
less likely that either therapeutic failure or toxicity will occur when the new
chemical entity is administered to humans or animals.
4.4 Graphical Kinetic Plots
Though less frequently used given the advent of personal computer programs
capable of performing nonlinear regression analysis of data quickly and
efficiently, the use of graphical plots in analyzing kinetic data can give quick
estimates of relevant kinetic parameters. In addition, graphical replots of
kinetic data can have diagnostic value in helping to deduce kinetic mechanisms
with multiple substrates or with inhibition (see below). Nonlinear regression
analysis of the data with the appropriate model (e.g., Michaelis–Menten
model) will most accurately generate estimates of enzyme kinetic parameters.
Probably the most commonly used graphical plot in analyzing enzyme
kinetic data is the double-reciprocal plot (Lineweaver–Burk plot) (Fig. 4.2). In
this analysis the Michaelis–Menten equation is rewritten so that the results can
be plotted as a straight line where 1/nis plotted along they-axis and 1/[S] along
thex-axis. In this plot, the slope of the line best fitting the data points is equal
toKm/Vmax, they-intercept = 1/Vmaxand thex-intercept =1/Km. It should be
noted that this type of plotting suffers from potentially large errors in 1/n.At
92 ENZYME KINETICS