CYP2E1 Metabolism of Styrene Involves Allostery

Abstract

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 (K s ‫؍‬ 830 M). A second styrene molecule then binds CYP2E1 with higher affinity (K ss ‫؍‬ 110 M) and significantly improves oxidation to achieve a k cat of 6.3 nmol ⅐ min ؊1 ⅐ 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 K i 0.51 to K si 0.043 M. The inhibitor was a negative allosteric effector on styrene oxidation, because k cat decreased 6-fold to 0.98 nmol ⅐ min ؊1 ⅐ 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.

Structural basis for cooperative binding of azoles to CYP2E1 as interpreted through guided molecular dynamics simulations

“…For 4-nitrophenol, metabolic rates of turnover increase and then decrease as a function of substrate concentration indicating substrate inhibition [3, 6]. Alternatively, many CYP2E1 substrates, including phenacetin, m -xylene [5], styrene [7, 8], and 7-ethoxycoumarin, demonstrate a poor efficiency in turnover at low substrate concentrations that rapidly improves at higher concentrations through a positive cooperative mechanism. Recent studies have further shown that aniline metabolism by CYP2E1 metabolism involves negative cooperativity in which higher substrate concentrations inhibit the ability for the enzyme to reach a maximal rate [9].…”

The genetic consequences of paternal acrylamide exposure and potential for amelioration

“…Resveratrol does demonstrate the potential for combatting acrylamide induced DNA damage via Cyp2e1 inhibition. A number of other CYP2E1 inhibitors have been identified using the human enzyme and these warrant further investigation (and potential modification) to assess their ability to inhibit the effects of acrylamide [100][101][102]. Future clinical application of natural or modified CYP2E1 inhibitors may be of benefit to those males ingesting acrylamide while attempting fertilisation.…”

Cooperativity in CYP2E1 metabolism of acetaminophen and styrene mixtures

“…2 Kinetic profiles for CYP2E1 substrates are often hyperbolic and thus best fit to the Michaelis-Menten equation in which the metabolic efficiency is constant. Unlike traditional enzymes, a more complex array of mechanistic possibilities are possible for CYP2E1 based on recent steady-state studies on toxicologically important substrates including aniline, 3 styrene, 4 and m -xylene. 5 The metabolism of those pollutants involved non-hyperbolic kinetic profiles, whereby metabolic efficiency either increased (positive cooperativity) or decreased (negative cooperativity or substrate inhibition) as a function of substrate concentration.…”

“…Those kinetic profiles could be explained through mechanisms involving catalytic and effector sites for CYP2E1. 3, 4, 6–11 Their presence and specificities were further characterized through experimental catalytic inhibition 6, 8, 12 and binding 6, 8 studies and computational docking and Molecular Dynamics simulations 11 using a series of mono- and bi-cyclic azoles. A hallmark of these studies has been the capacity of substrates alone and combined with inhibitors to impact CYP2E1 metabolic efficiency in ways unexpected by the traditional Michaelis-Menten mechanism.…”

“…The resulting data were global fitting to six possible mechanisms involving a single binding site (Michaelis-Menten) or two binding sites and the most statistically probable one identified using DynaFit software 24 as described. 3, 4, 6 Lastly, we assessed the potential impact of acetaminophen on the predicted in vivo metabolic activation of styrene by CYP2E1.…”