Cytochrome P450 (CYP) enzymes facilitate the oxidation of xenobiotic compounds, contributing significantly to their pharmacokinetics. One isoform in particular, Cytochrome P450 3A4 (CYP3A4), is thought to be responsible for over 50% of oxidative drug metabolism because of high expression levels in the gut and the liver and a high level of substrate promiscuity. CYP3A4 in vitro allosterism remains unexplained from a mechanistic standpoint and complicates in vitro to in vivo scaling. Using the archetypal allosteric effector molecule alphanapthoflavone (ANF) we report the kinetic parameters for the sequential de-ethylation of fluorescent substrate Nile Red (NR) to its mono and di des-ethyl metabolites M1 and M2. ANF increases Vmax for NR metabolism to both products while the Km is not significantly affected. ANF also decreases uncoupling at the point of peroxide production, indicating that increased coupling in the P450 cycle is a probable mechanism for the observed heterotropic allosteric activation.

Exploring Alphanapthoflavone heterotropic allosteric regulation of Cytochrome P450 3A4

240th National Meeting (2010)

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