Figure 1
P450 probes and reaction mechanisms. (a) Structures of five activity-based probes for P450s. All probes contain an alkyne minitag (right, blue circles), for coupling to azide-fluorophores or azide-biotin via click chemistry. The reactive group (left, red rectangles) is an aryl alkyne-based reactivity group, except for DB080, which contains a furan reactivity group. (b) Labeling mechanisms. The P450 probes are oxygenated by P450, leading to a reactive intermediate that covalently links to nearby residues in the substrate binding site of P450s. (c) Click chemistry with picolyl azide. The coupling of the P450-alkyne to azide-tag is catalyzed by copper(I), which is stabilized by the ligand (tris((1-benzyl-1H-1,2,3-triazol-4-yl) methyl)amine, TBTA), the picolyl, and the azide, resulting in a stable triazole linker between the P450 protein and a reporter tag. The reporter tag is either biotin or a fluorophore.
