Functioning of drug-metabolizing microsomal cytochrome P450s: In silico probing of proteins suggests that the distal heme 'active site' pocket plays a relatively 'passive role' in some enzyme-substrate interactions.

Avanthika Venkatachalam,Kelath Murali Manoj,Abhinav Parashar

doi:10.1186/s40203-016-0016-7

Avanthika Venkatachalam, Kelath Murali Manoj + Show 1 more

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https://doi.org/10.1186/s40203-016-0016-7

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Abstract

PurposeThe currently held mechanistic understanding of microsomal cytochrome P450s (CYPs) seeks that diverse drug molecules bind within the deep-seated distal heme pocket and subsequently react at the heme centre. To explain a bevy of experimental observations and meta-analyses, we indulge a hypothesis that involves a “diffusible radical mediated” mechanism. This new hypothesis posits that many substrates could also bind at alternate loci on/within the enzyme and be reacted without the pertinent moiety accessing a bonding proximity to the purported catalytic Fe-O enzyme intermediate.MethodsThrough blind and heme-distal pocket centered dockings of various substrates and non-substrates (drug molecules of diverse sizes, classes, topographies etc.) of microsomal CYPs, we explored the possibility of access of substrates via the distal channels, its binding energies, docking orientations, distance of reactive moieties (or molecule per se) to/from the heme centre, etc. We investigated specific cases like- (a) large drug molecules as substrates, (b) classical marker drug substrates, (c) class of drugs as substrates (Sartans, Statins etc.), (d) substrate preferences between related and unrelated CYPs, (e) man-made site-directed mutants’ and naturally occurring mutants’ reactivity and metabolic disposition, (f) drug-drug interactions, (g) overall affinities of drug substrate versus oxidized product, (h) meta-analysis of in silico versus experimental binding constants and reaction/residence times etc.ResultsIt was found that heme-centered dockings of the substrate/modulator drug molecules with the available CYP crystal structures gave poor docking geometries and distances from Fe-heme centre. In conjunction with several other arguments, the findings discount the relevance of erstwhile hypothesis in many CYP systems. Consequently, the newly proposed hypothesis is deemed a viable alternate, as it satisfies Occam’s razor.ConclusionsThe new proposal affords expanded scope for explaining the mechanism, kinetics and overall phenomenology of CYP mediated drug metabolism. It is now understood that the heme-iron and the hydrophobic distal pocket of CYPs serve primarily to stabilize the reactive intermediate (diffusible radical) and the surface or crypts of the apoprotein bind to the xenobiotic substrate (and in some cases, the heme distal pocket could also serve the latter function). Thus, CYPs enhance reaction rates and selectivity/specificity via a hitherto unrecognized modality.Electronic supplementary materialThe online version of this article (doi:10.1186/s40203-016-0016-7) contains supplementary material, which is available to authorized users.

Highlights

The cytochrome P450 (CYP) family of enzymes possesses the heme-thiolate functionality and they mediate the phase I metabolism of a vast majority of drugs and xenobiotics in most animals, including man (Testa 1995)
It is understood that the heme-iron and the hydrophobic distal pocket of CYPs serve primarily to stabilize the reactive intermediate and the surface or crypts of the apoprotein bind to the xenobiotic substrate
Distal heme active site cavities/tunnels of some CYPs and interactions of the enzyme with substrates Table 2 details the physical dimensions of- active site cavity, classical substrates and the tunnels from distal heme centre leading to the solvent continuum

Summary

Introduction

The cytochrome P450 (CYP) family of enzymes possesses the heme-thiolate functionality and they mediate the phase I metabolism of a vast majority of drugs and xenobiotics in most animals, including man (Testa 1995). Many of these reactions are known to be regiospecific and some of them are even enantioselective (Martinez and Stewart 2000). The fundamental step in the catalytic mechanism invoked the formation of high potential intermediate(s) centered at the heme-thiolate moiety, involving an iron - oxygen species (Ortiz de Montellano 2015; Meunier et al 2004; Denisov et al 2005; Volz et al 2002). When the substrate gets converted to the product, the latter is supposed to lose affinity for the enzyme and it detaches and diffuses out of the ‘active site’

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