Origin of Selectivity in Protein Hydrolysis by Zr(IV)-Containing Metal Oxides as Artificial Proteases

© 2020 American Chemical Society. All rights reserved. The origin of selectivity in protein hydrolysis promoted by Zr(IV)-substituted polyoxometalates (POMs) has been investigated through a variety of computational techniques. Initially, we analyzed the reaction mechanism for the observed hydrolysis at the Asn44 - Arg45 site in the hen egg-white lysozyme protein (HEWL) by the Zr-substituted Lindqvist anion [W5O18Zr(H2O)(OH)]3- (ZrL) using cluster models obtained from molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, and metadynamics simulations. The mechanism characterization shows that the overall activity is governed by the energy cost to reach the transition state for C-N bond cleavage from the reactants, resulting in a calculated overall free-energy barrier of 121 kJ mol-1 that is in excellent agreement with the values derived from the experimental rate constants (113-134 kJ mol-1). In addition, QM/MM metadynamics simulations on the early stages of the mechanism revealed the formation of an exergonic non-covalent POM···protein complex at the protein surface that was stabilized by positively charged amino acids maintained during the Zr coordination to the amide oxygen. For nonreactive related sites containing Arg (Asn113 - Arg114, Arg45 - Asn46, and Arg21 - Gly22,) we found very similar overall barriers within the cluster model approach (124, 124, and 120 kJ mol-1, respectively); however, their nonbonding POM···protein interactions along the simulated coordination of Zr to the amide oxygen were significantly weaker than those for the reactive Asn44 - Arg45 site. Thus, for the HEWL protein the selectivity is governed by an enzyme-like recognition of ZrL at the cleavage site that results in an overall acceleration of the reaction rate compared to those at other sites. Conversely for human serum albumin, (HSA) the observed selectivity was not directed by nonbonding POM···protein interactions but instead was controlled by the protein secondary structure. Calculations on several Arg - Leu sites placed in positive patches showed that peptide bonds in an α-helix structure have higher overall free-energy barriers, while for the active Arg114 - Leu115 site in a random coil region the C-N cleavage is facilitated by the extended conformation of the protein chain. All in all, this study has identified and evaluated two complementary factors controlling the selectivity in peptide hydrolysis promoted by transition metal-substituted POMs; hydrolysis is disfavored at α-helical regions of the protein, and then specific positively charged patches can trap the POM via electrostatic-type POM···protein interactions and accelerate the reaction