Drug Metabolism in Drug Design and Development Basic Concepts and Practice

The apparentVmaxandKmwere determined to be 42.5 nmol/min/mg protein

and 180.0mM, respectively, indicating a high capacity with a moderate affinity

reaction. To ensure the suitability and accuracy of the parameter determination

provided by the nonlinear regression analyses, the traditional biochemical plots,

such as Michaelis–Menten or Eadie–Hofstee plots, can be used (Fig. 13.2).

Biphasic kinetics should preferably be analyzed using such a computational

approach. The above mathematic model can be revised to comprise two

independent hyperbolic components:y=ax/(b+x)+cx/(d+x). Hereaandb

are, respectively,VmaxandKmfor one kinetic component, andcanddare those

for the other, respectively. After the raw data (SandV), used for the construction

of the Michaelis–Menten plot shown in Fig. 13.3, were processed using

SigmaPlot, following the procedure as described, the results for the nonlinear

regression analyses were generated, they are summarized in Table 13.5. Of the

TABLE 13.4 Results of nonlinear regression analysis forKmandVmax

determination.

Equation: Hyperbola (y=ax/(b+x))
rr^2 ab
0.999 0.999 42.5 (P<0.0001) 180.0 (P<0.0001)
Regression
DF SS MS FP
1 637.1 637.1 3635.5 <0.0001
Statistical test
Normality Durbin–Watson Constant variance
Pass Pass Pass
Kinetic parameter
Kmapp(mM) Vmaxapp(nmol/min/mg)
180.0 42.5

TABLE 13.5 Results of nonlinear regression analyses forKmandVmax

determination with two enzymes involved in the catalysis.

Equation: Hyperbola with two components (y=ax/(b+x)+cx/(d+x))

rr^2 abc d

0.999 0.999 17.5

(P<0.0001)

0.46

(P<0.0001)

65.1

(P<0.0001)

24.5

(P<0.0001)

Regression

DF SS MS FP

3 2079.2 693.1 1697.0 <0.0001

Statistical test

Normality Durbin–Watson Constant variance

Pass Pass Pass

Kinetic parameter

Kappm (mM) Vmaxapp(nmol/min/mg)

0.46 24.5 17.5 65.1

CHARACTERIZATION OF ENZYME KINETICS 431