2001). For instance, six, or preferably eight, concentrations, covering a range
of 0–4 mM, could be considered for the initiation of the study. These
concentrations could be 0, 1, 10, 50, 200, 500, 1000, and 4000mM. The range of
substrate concentrations is adjustable, and can be narrowed ifKmappcan be
approximated. However, for accurate calculations, the metabolic rates
generated at the lower substrate concentration (i.e., 1mM), or the higher
substrate concentrations (i.e., 4 mM), may have to be disregarded if, during the
incubation, the lower concentrations are markedly changed, or if substrate
inhibition, a common form of atypical kinetics, is observed at the higher
concentrations. Therefore, if Kappm is presumed to fall within the range of
20–400mM, the above substrate concentration selection could be revised to 0,
10, 20, 50, 100, 200, 500, and 1200mM.
13.3.3 Determination of Kinetic Parameters
13.3.3.1 Biochemical Plots Several methods are readily applied to the
determination of kinetic parameters (KmandVmax). Traditionally, these terms
are determined using the classic biochemical plots, particularly those
transformed from the well-known Michaelis–Menten plot, for example,
Lineweaver–Burk and Eadie–Hofstee plots (Li et al., 1995; Nakajima et al.,
2002; Nnane et al., 2003; Yamamoto et al., 2003).
Lineweaver–Burk plots have been widely used in biochemical studies,
although their intrinsic limitations are occasionally overlooked. For instance,
as shown in Fig. 13.2, the data points in Lineweaver–Burk plots tend to be
unevenly distributed, thus potentially leading to unreliable reciprocals at lower
metabolic rates (1/V). These lower rates dictate the linear regression curves and,
therefore, the apparent values ofKmandVmax. In contrast, the data points in
Eadie–Hofstee plots are usually homogeneously distributed, and thus tend
to be more accurate. Eadie–Hofstee plots are diagnostic for biphasic kinetics
that arises from the involvement of multiple enzymes having differing
kinetic properties, or from atypical kinetics, such as homotropic cooperation
(Fig. 13.3) (Zhang and Kaminsky, 1995; Zhang and Wong, 2005). One example
is theophylline 8-hydroxylation, the major primary metabolic pathway of
theophylline in humans (Campbell et al., 1987; Zhang and Kaminsky, 1995).
The kinetics of theophylline 8-hydroxylation in a HLM system appeared
biphasic (Campbell et al., 1987). Multiple CYPs were implicated in the
metabolism, including CYP1A2, CYP2D6, CYP2E1, and CYP3A4, character-
ized by markedly differing substrate binding affinities, as indicated by theKappm
values: 0.6 mM for CYP1A2, but more than 10 mM for the others (Zhang and
Kaminsky, 1995). The intrinsic clearance (CLint) estimated byVmax/Kmfor the
individual CYP enzymes, and the correlation analyses of the CYP form-specific
activities with theophylline 8-hydroxylation activity at 5 and 40 mM of
theophylline using a panel of HLM preparations, confirmed the primary role of
CYP1A2 in this major biotransformation of theophylline in humans. There
are, however, potential contributions from the other species, CYP2E1, and
CHARACTERIZATION OF ENZYME KINETICS 427