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 μM). A second styrene molecule then binds CYP2E1 with higher affinity (K_ss = 110 μM) and significantly improves oxidation to achieve a k_cat 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 K_i 0.51 to K_si 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because k_cat 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.