Figure 1.

A) Simplified catalytic mechanism of cysteine and serine proteases (Nu=S, e.g., in TEV^Cys; or Nu=O, e.g., in TEV^Ser).⁷ In the enzyme’s catalytic triad (black) aspartate aligns and polarises histidine, which reduces the nucleophile’s pK_a and positions it for reaction. The activated nucleophile attacks the carbonyl of the peptide bond of the substrate (red), a tetrahedral intermediate is stabilised by backbone amide hydrogens (“oxyanion hole”), and the C terminus of the substrate is ejected, leaving a covalent adduct behind. This acyl–enzyme intermediate (lower panel) is hydrolysed by histidine-activated water to release the N terminus of the substrate (aided by proton transfer to histidine), and this regenerates the free enzyme. B) The phylogeny of the PA clan (MEROPS classification)¹¹ of proteases with nucleophile and possible nucleophile-switching events indicated. Proteases with known structures were aligned to TEV^Cys by structural comparison using DALI,¹³ sequences of unknown structure were added by sequence alignment with BLASTp (see Table S1 and Figure S1 for details).