The cysteine protease from the tobacco etch virus (TEVp) is a well-known and widely utilized enzyme. TEVp's chymotrypsin-like fold is generally associated with serine catalytic triads that differ in terms of a reaction mechanism from the most well-studied papain-like cysteine proteases. The question of what dominates the TEVp mechanism, nucleophile identity, or structural composition has never been previously addressed. Here, we use enhanced sampling multiscale modeling to uncover that TEVp combines the features of two worlds in such a way that potentially hampers its activity. We show that TEVp cysteine is strictly in the anionic form in a free enzyme similar to papain. Peptide binding shifts the equilibrium toward the nucleophile's protonated form, characteristic of chymotrypsin-like proteases, although the cysteinyl anion form is still present and interconversion is rapid. This way cysteine protonation generates enzyme states that are a diversion from the most effective course of action, with only 13.2% of Michaelis complex sub-states able to initiate the reaction. As a result, we propose an updated view on the reaction mechanism catalyzed by TEVp. We also demonstrate that AlphaFold is able to construct protease-substrate complexes with high accuracy. We propose that our findings open a way for its industrious use in enzymological tasks. Unique features of TEVp discovered in this work open a discussion on the evolutionary history and trade-offs of optimizing serine triad-associated folds to cysteine as a nucleophile.
© 2022 The Authors. Published by American Chemical Society.