Xanthates As Useful Probes for Testing the Active Sites of Cytochromes P450 4A11 and 2E1.

Tsveta Stoyanova,Ilza Pajeva,Momir Mikov,Stanislav Yanev,Iglika Lessigiarska

doi:10.3389/fphar.2017.00672

Abstract

Xanthates (alkyl or aryl derivatives of dithiocarbonic acid) have been shown to be selective mechanism-based inactivators of cytochromes P450 2B1/2B6 and 2E1 due to covalent binding of a reactive intermediate to apoprotein after double hydrogen abstraction at α-carbon atom, suggesting interaction of the xanthate dithiocarbonic head with the enzyme heme. The structures of xanthates with a long alkyl chain are similar to the fatty acids. Saturated fatty acids (FA) such as lauric acid (LA), are metabolized by different cytochrome P450 isoforms to ω- and (ω-1)-hydroxy products, in humans done by CYP4A11 and CYP2E1, respectively. In the present study we aimed at elucidating the possible interactions of xanthates with two cytochrome P450 isoforms CYP4A11 and CYP2E1 involved in the metabolism of the FA. Our experiments showed that LA-ω-hydroxylation by CYP4A11 is inhibited in a competitive manner by xanthates with long alkyl chain (C12-xanthate being the most potent inhibitor). On the other hand LA-(ω-1)-hydroxylation reaction by purified CYP2E1 is inactivated by a mechanism-based type. The suggested differences in the interactions of C12-xanthate with the two cytochrome P450 isoforms were investigated by molecular modeling using docking approach. The results suggested that in CYP2E1 active site C12-xanthate coordinates to the heme with its most vulnerable dithiocarbonic head leading to a mechanism-based inactivation. In CYP4A11 xanthate alkyl chain is exposed to the heme, thus, a potenial ω-hydroxylated xanthate product could be formed, which could inhibit in a competitive manner the hydroxylation of LA. The observed differences of xanthates interactions with the active sites of the two similar cytochrome P450 isoforms (CYP4A11 and CYP2E1) involved in the metabolism of FA, which lead to different changes in the enzyme activity, suggest that xanthates can be used as probing tools for analyzing enzyme active sites when exploring useful and selective compounds influencing FA homeostasis.

Highlights

Fatty acids metabolism is related to many physiological and pathological processes like diabetes, gene expression (Jump et al, 1999), lipid peroxidation (Gueraud et al, 1999), regulation of vascular tone (Nguyen et al, 1999), immune system (Calder, 1999), hypertension (Engler et al, 1999), inflammation and cancer (Johnson et al, 2015).Saturated fatty acids (FA) such as lauric acid (LA), are metabolized by Cytochrome P450 (CYP)-dependent reactions to ω- and (ω-1)-hydroxy products
Enzyme activity tends to increase with fatty acid chain length [the activity is higher for myristic (C10) and lauric (C12) acids] and the ratio of ω-hydroxylase to (ω-1)-hydroxylase activity dependents on the particular substrate and P450 isozymes
The hydroxylation of LA was affected by the xanthates in a different manner depending on the CYP isoform involved in the reaction

Summary

Introduction

Saturated FAs such as LA, are metabolized by CYP-dependent reactions to ω- and (ω-1)-hydroxy products. Enzyme activity tends to increase with fatty acid chain length [the activity is higher for myristic (C10) and lauric (C12) acids] and the ratio of ω-hydroxylase to (ω-1)-hydroxylase activity dependents on the particular substrate and P450 isozymes. The CYP isozymes involved in the FAs metabolism are mainly from 4A to 2E families. They have different species and organ distribution: human 4A9, 4A11, 4A12, 4F2 (liver and kidney) and 2E1 forms, rat 4A1, 4A2, 4A3, and 4A8 forms (liver, kidney, and brain) (Roman, 2002). The human liver 4F2 is mainly involved in arachidonic acid ω-hydroxylation (Powell et al, 1998)

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