A comparison of substrate dynamics in human CYP2E1 and CYP2A6

Abstract

Considering the dynamic nature of CYPs, methods that reveal information about substrate and enzyme dynamics are necessary to generate predictive models. To compare substrate dynamics in CYP2E1 and CYP2A6, intramolecular isotope effect experiments were conducted, using deuterium labeled substrates: o-xylene, m-xylene, p-xylene, 2,6-dimethylnaphthalene, and 4,4′-dimethylbiphenyl. Competitive intermolecular experiments were also conducted using d 0 -and d 6 -labeled p-xylene. Both CYP2E1 and CYP2A6 displayed full isotope effect expression for o-xylene oxidation and almost complete suppression for dimethylbiphenyl. Interestingly, (k H /k D ) obs for d 3 -p-xylene oxidation ((k H /k D ) obs = 6.04 and (k H /k D ) obs = 5.53 for CYP2E1 and CYP2A6, respectively) was only slightly higher than (k H /k D ) obs for d 3 -dimethylnaphthalene ((k H /k D ) obs = 5.50 and (k H / k D ) obs = 4.96, respectively). One explanation is that in some instances (k H /k D ) obs values are generated by the presence of two substrates bound simultaneously to the CYP. Speculatively, if this explanation is valid, then intramolecular isotope effect experiments should be useful in the mechanistic investigation of P450 cooperativity. KeywordsCYP2E1; CYP2A6; Isotope effect; Cooperativity; Allosterism; Dynamics; P450; k H /k D ; Xylene Some Cytochromes P450 (CYP or P450) catalyze the oxidative metabolism of exogenous compounds, including drugs, to metabolites that are usually less toxic and more water soluble, especially after conjugation. This process is important since it reduces the risk of drug-induced toxicity by facilitating drug elimination. However, in certain cases, CYP-catalyzed oxidation may also lead to the formation of reactive, electrophilic metabolites that can lead to toxicity or carcinogenicity. Inhibition or induction of specific P450 isoforms can also be a source of drug-drug interactions. On account of these important roles that CYPs play in drug metabolism and drug toxicity, a substantial effort to characterize the drug-metabolizing CYPs continues with hopes of predicting such details as isoform selectivity of new drug candidates, rates of metabolite formation, product selectivity, and the formation of toxic metabolites.The development of accurate predictive models requires, among other things, an understanding of structure (both of the enzyme and the substrate), mechanism, and kinetics. With CYP

Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes

“…However, analogous studies with deuterated 4,4′-dimethylbiphenyl showed that the hydroxylation isotope effect in the oxidation of this substrate, in which the methyl groups are separated by a distance of 11.05 Å, is almost completely masked. Similar results were obtained when the studies were extended to CYP2E1 and CYP2A6: the ( k H / k D ) obs values for CYP2E1 were 9.03 for o -xylene, 6.65 for m -xylene, 6.04 for p -xylene, and 2.28 for 4,4′-dimethylbiphenyl; for CYP2A6 the corresponding values were 11.46, 7.21, 5.53, and 1.07 (108). A follow up experiment with CYP101, for which a crystal structure is available, gave observed isotope effects for the substrates with one trideuterated methyl group of 10.6 for o -xylene, 7.4 for p -xylene, and 2.7 for 4,4′-dimethylbiphenyl (109).…”

Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole

“…Our results indicate a metabolism-mediated interaction with cinnamon or t-CA would be markedly more probable for CYP2A6 substrates than for drugs metabolized by the other P450 isoforms typically evaluated during drug discovery (Food and Drug Administration, 2012). Even though CYP2A6 and CYP2E1 exhibit overlap in substrate selectivity (Yoo et al, 1990;Yamazaki et al, 1992;Chen et al, 1998;Harrelson et al, 2007), t-CA was 10.5-fold more selective for CYP2A6. The IC 50 values for other major drug-metabolizing P450s were at least 16-fold higher or more, and in some cases, substantially higher (i.e., 2D6 and 2C19).…”

Cooperative properties of cytochromes P450

“…Interactions of CYP2E1 with two molecules of substrate p-nitrophenol or with one substrate and one inhibitor molecule results in non-Michaelis kinetics with low rates of product formation at high substrate concentrations (Collom et al, 2008). Binding of two substrate molecules by CYP2A6 and CYP2E1 was also inferred by the intramolecular isotope effects of hydroxylation of several aromatic substrates (Harrelson et al, 2007). Later, oxidation of m -xylene (cooperative) and p -xylene (non-cooperative), were studied using selectively deuterated substrates (Harrelson et al, 2008).…”