Abstract
We are the first to report allosterism during styrene oxidation by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1 (Ks = 830 μM). A second styrene molecule then binds CYP2E1 with higher affinity (Kss = 110 μM) and significantly improves oxidation to achieve a kcat of 6.3 nmol · min−1 · nmol CYP2E1−1. The transition between these metabolic cycles coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Scaled modeling of the in vitro-positive allosteric mechanism for styrene metabolism to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low styrene levels were notably much less toxic than generally assumed. We interrogated the allosteric mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. From the current studies, styrene was a positive allosteric effector on 4-methylpyrazole binding, based on a 10-fold increase in 4-methylpyrazole binding affinity from Ki 0.51 to Ksi 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because kcat decreased 6-fold to 0.98 nmol · min−1 · nmol CYP2E1−1. Consequently, mixtures of styrene and other molecules can induce allosteric effects on binding and metabolism by CYP2E1 and thus mitigate the efficiency of their metabolism and corresponding effects on human health. Taken together, our elucidation of mechanisms for these allosteric reactions provides a powerful tool for further investigating the complexities of CYP2E1 metabolism of drugs and pollutants.
Footnotes
This work was supported in part by the National Institutes of Health National Center for Research Resources [Grant P20-RR-16460] (IDeA Networks of Biomedical Research Excellence Program; to J.H.H. and G.P.M.); the Summer Undergraduate Research Fellowship sponsored by the Biochemistry and Molecular Biology Department at the University of Arkansas for Medical Sciences (to J.H.H. and G.P.M.); a bridging grant from the University of Arkansas for Medical Sciences (to G.P.M.); and the Arkansas Tobacco Settlement Proceeds Act of 2000 (to G.B).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- P450
- cytochrome P450
- HLM
- human liver microsomes
- HPLC
- high-performance liquid chromatography
- ES
- enzyme-substrate
- ESS
- enzyme-substrate-substrate
- ESI
- enzyme-substrate-inhibitor
- AIC
- Akaike's information criterion.
- Received May 15, 2012.
- Accepted July 17, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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