phenomenon is thought to occur due to simultaneous binding of both substrate
and effector molecule within the enzyme active site simultaneously. One of the
first conclusive demonstrations of this phenomenon was the observation that
the CYP3A4 substrate 7,8-benzoflavone increased theVmaxof phenanthrene
metabolism without changing theKmand conversely, phenanthrene decreased
theVmaxof 7,8-benzoflavone metabolism, again with no effect onKm(Shou
et al., 1994). Later, it was shown that dapsone increased theVmaxand decreased
theKmof CYP2C9-mediated flurbiprofen metabolism, while only minimal
changes in dapsone metabolism were noted (Hutzler et al., 2001). Numerous
examples of heterotropic activation of cytochrome P450 and glucuronosyl
transferase mediated metabolism, as well as P-glycoprotein transport have
been noted (Egnell et al., 2003; Hutzler and Tracy, 2002; Kenworthy et al.,
2001; Pfeiffer et al., 2005; Taub et al., 2005; Witherow and Houston, 1999).
In the simplest case, this activation is of a nonessential nature and maintains
the hyperbolic shape of the kinetic profile (Fig. 4.8). ThoughKmandVmaxmay
change, the velocity versus substrate curve remains hyperbolic.
Certainly, one can fit each of the individual velocity versus substrate curves
at each effector concentration and determine multipleKms andVmaxs for the
process of activation. However, it may be more informative to fit all the data
together to ascertain additional information regarding the phenomenon. In this
case, equation (Eq. 4.11) may be used to estimateKm andVmax for the
FIGURE 4.8 Representative plot depicting heterotropic cooperativity (activation
kinetics) where velocity of the reaction increases with increasing effector (activator)
concentration.
100 ENZYME KINETICS