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An NMR‐Based Binding Assay Used To Screen For Compounds That Bind To Cytochrome P450 Enzymes
Author(s) -
McCullough Christopher Robert,
Pullela Phani Kumar,
Sem Daniel S.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a939-d
Subject(s) - chemistry , heme , heteronuclear single quantum coherence spectroscopy , enzyme , cytochrome p450 , chemical shift , stereochemistry , cytochrome , ligand (biochemistry) , moiety , biochemistry , active site , nuclear magnetic resonance spectroscopy , receptor
The cytochromes P450 are the enzymes predominantly responsible for the metabolism of drugs and other exogenous compounds that enter into our bodies. Because of these enzymes’ prominent role, it would be helpful to be able to quickly predict which compounds—potential drug candidates, toxins, or protoxins—bind to these enzymes, thus providing invaluable data to serve as the starting point for later, more thorough studies. This quick prediction can be accomplished in the form of a high‐throughput assay that involves monitoring the 13 C‐attached 1 H chemical shift ( 13 C‐attached 1 H δ) and 13 C δ of 13 CH 3 NC (methyl isocyanide). As is the case for any short‐chain alkylisocyanide, methyl isocyanide binds through its isonitrile moiety to the heme iron contained within the active site of the cytochrome P450 enzymes. We show here, in a series of 13 C‐ 1 H HSQC spectra and for several cytochrome P450s, that the two previously mentioned chemical shifts of this ligand change in going from the free (unbound) form to the bound form and then, again upon addition of a known substrate (distinct from both bound and unbound). In addition, for the two full‐length, membrane‐bound P450s, we show that the chemical shifts indicative of the bound form change depending on whether the protein has been solubilized in 20% (v/v) glycerol or incorporated into DPC micelles.