CYP2E1 hydroxylation of aniline involves negative cooperativity.

Area of Science:

• Biochemistry
• Pharmacology
• Enzyme kinetics

Background:

• Cytochrome P450 2E1 (CYP2E1) is implicated in aniline metabolism, but its precise mechanism and relevance remain unclear.
• Conflicting reports exist regarding the role and kinetics of CYP2E1 in aniline metabolic activation and elimination.

Purpose of the Study:

• To investigate the hypothesis that aniline metabolism by CYP2E1 involves two binding sites, leading to cooperative kinetics.
• To elucidate the mechanistic details of aniline-CYP2E1 interactions and their implications for in vivo clearance.

Main Methods:

• In vitro kinetic studies using recombinant CYP2E1 and human liver microsomes.
• Analysis of kinetic data using the Hill equation and a two-binding site cooperative model.
• Inhibition studies with 4-methylpyrazole and inhibitor phenotyping experiments.

Main Results:

• Recombinant CYP2E1 demonstrated significant negative cooperativity (n=0.56) in aniline metabolism.
• A two-binding site model explained the data, with high-affinity (K(s)=30 μM) and lower-affinity (K(ss)=1100 uM) binding sites.
• CYP2E1 was confirmed as the primary enzyme, with minor contributions from CYP2A6 and CYP2C9.
• Inhibition of minor pathways replicated CYP2E1 kinetics, validating the cooperative model.

Conclusions:

• Aniline metabolism by CYP2E1 exhibits negative cooperativity, a novel finding for this enzyme-CYP450 interaction.
• The identified cooperative binding mechanism significantly impacts scaled modeling of in vivo aniline clearance, especially at low concentrations.
• This study provides critical mechanistic insights into CYP2E1's role in aniline metabolism and elimination.