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Arch Biochem Biophys. 2011 Mar 1;507(1):135-43. doi: 10.1016/ Epub 2010 Sep 15.

Targeting of the highly conserved threonine 302 residue of cytochromes P450 2B family during mechanism-based inactivation by aryl acetylenes.

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Department of Pharmacology, The University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.


Cytochromes P450 (CYPs or P450s) contain a highly conserved threonine residue in the active site, which is referred to as Thr302 in the amino acid sequence of CYP2B4. Extensive biochemical and crystallographic studies have established that this Thr302 plays a critical role in activating molecular oxygen to generate Compound I, a putative iron(IV)-oxo porphyrin cation radical, that carries out the preliminary oxygenation of CYP substrates. Because of its proximity to the center of the P450 active site, this Thr302 is susceptible to mechanism-based inactivation under certain conditions. In this article, we review recent studies on the mechanism-based inactivation of three mammalian P450s in the 2B family, CYP2B1 (rat), 2B4 (rabbit) and 2B6 (human) by tert-butylphenylacetylene (tBPA). These studies showed that tBPA is a potent mechanism-based inactivator of CYP2B1, 2B4 and 2B6 with high k(inact)/K(I) ratios (0.23-2.3min(-1)μM(-1)) and low partition ratios (0-5). Furthermore, mechanistic studies revealed that tBPA inactivates these three CYP2B enzymes through the formation of a single ester adduct with the Thr302 in the active site. These inhibitory properties of tBPA allowed the preparation of a modified CYP2B4 where the Thr302 was covalently and stoichiometrically labeled by a reactive intermediate of tBPA in quantities large enough to permit spectroscopic and crystallographic studies of the consequences of covalent modification of Thr302. Molecular modeling studies revealed a unique binding mode of tBPA in the active site that may shed light on the potency of this inhibition. The results from these studies may serve as a basis for designing more specific and potent inhibitors for P450s by targeting this highly conserved threonine residue which is present in the active sites of most mammalian P450s.

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