Herein, we investigate the structure and flexibility of the hydrated SARS-CoV-2 main protease by means of 2.0 μs molecular dynamics (MD) simulations in explicit solvent. After having performed electrostatic p K a calculations on several X-ray structures, we consider both the native (unbound) configuration of the enzyme and its noncovalent complex with a model peptide, Ace-Ala-Val-Leu-Gln∼Ser-Nme, which mimics the polyprotein sequence recognized at the active site. For each configuration, we also study their monomeric and homodimeric forms. The simulations of the unbound systems show that the relative orientation of domain III is not stable in the monomeric form and provide further details about interdomain motions, protomer–protomer interactions, inter-residue contacts, accessibility at the catalytic site, etc. In the presence of the peptide substrate, the monomeric protease exhibits a stable interdomain arrangement, but the relative orientation between the scissile peptide bond and the catalytic dyad is not favorable for catalysis. By means of comparative analysis, we further assess the catalytic impact of the enzyme dimerization, the actual flexibility of the active site region, and other structural effects induced by substrate binding. Overall, our computational results complement previous crystallographic studies on the SARS-CoV-2 enzyme and, together with other simulation studies, should contribute to outline useful structure–activity relationships.

Quantum chemical optimizations of the small model systems ([Zn(NH 3 ) ) were performed at different levels of quantum mechanical theory (HF/6-31G*, B3LYP/6-31G*, and MP2/6-31G*) to characterize the Znligand bonds for the Zn1 and Zn2 binding sites of metallo-β-lactamases. The nature of the zinc coordination environment was further studied by considering larger mononuclear complexes at the B3LYP/6-31G*//HF/ 6-31G* level of theory ([Zn(Me-Im) ). The structure and properties of a series of binuclear model compounds showing an hydroxy-mediated Zn1‚‚‚Zn2 interaction were also analyzed at the same level of theory. One of the binuclear models with a global charge of +2, reproduces the main structural features of the Bacteroides fragilis active site as determined by X-ray crystallography. The proposed β-lactamase model has a monoprotonated state characterized by a strong H-bond interaction between a zinc-shared water molecule and a Zn2-bound Asp carboxylic group. The theoretical results are discussed in the context of experimental kinetic and structural data on the B. fragilis active site, resulting in insights into the nature of the zinc-ligand interactions, the location of the mechanistically relevant water molecules, and the actual protonation state of the active site. By combining the present results with previous theoretical and experimental work, mechanistic details for the mode of action of zinc β-lactamases are discussed.

Herein, we present results from a computational study of dinickel complexes that are relevant to the catalytic hydrolysis of urea exerted by the urease enzymes. The B3LYP density functional is used to characterize the equilibrium geometry, electronic and magnetic properties, and energies for a series of realistic complexes modeling the active site of ureases. The analysis of the theoretical results gives new insight into the structure, substrate binding, and catalytic mechanism. The water bridge between the two Ni(II) ions observed in the crystallographic structures of the ureases was assigned to a hydroxide bridge in agreement with the observed small antiferromagnetic coupling. Both monodentate and bidentate urea-bound complexes, in which urea had favorable orientations for catalysis, were characterized. Finally, two reaction mechanisms were investigated starting from the monodentate and bidentate urea-bound complexes, respectively. Both a Ni1...Ni2 bridging hydroxide and a Ni2-bound water molecule play crucial roles in the two mechanisms.