A Combination of Computational and Experimental Approaches to Investigate the Binding Behavior of B.sub Lipase A Mutants with Substrate pNPP

Abstract

The formation of so-called enzyme-substrate complex is the key step for a successful enzyme-catalysis reaction. Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. Some residues besides the catalytic triads play important roles toward the substrate binding process. In this study, we employed ONIOM methodology and docking to explore the influence of individual amino acids of Bacillus subtilis (B.sub) lipase A on the hydrolysis reaction, with the aim to guide mutagenesis experiments on the basis of computational framework. Subsequently, the B.sub lipase A is modified experimentally with different non-polar residues at the position 12, which is spatially adjacent to the active site, by using site-directed mutagenesis. We obtain a good correlation model between the computationally predicted binding energies and the experimental measured affinities, with a correlation coefficient r=0.78. It is largely unexplored that the combination of docking and quantum mechanical/molecular mechanical (QM/MM) analyses is used in conjunction with experimental procedure to investigate the enzyme catalysis process. We therefore expect that this work could provide a new pathway for exploring the molecular mechanism of enzyme-substrate recognition and interaction.

Is Metal Stabilization of the Leaving Group Required or Can Lysine Facilitate Phosphodiester Bond Cleavage in Nucleic Acids? A Computational Study of EndoV

“…Based on our detailed analysis of the conformational dynamics of the EndoV active site, eight unique MD snapshots were extracted across the three MD simulation replicas to characterize the catalytic mechanism with QM/MM that differ in the combination of the general base and acid and metal coordination environment (Figure S6 and Table ). QM/MM calculations were performed using the ONIOM formulism due to this approach successfully characterizing the reaction pathways for many enzymes, including endonucleases. ,,− Regardless of the metal–substrate coordination geometry, the QM region included D43, D110, and two water molecules directly coordinated to Mg 2+ , H214, K139, E89, a nucleophilic water, the (dG229, dA230) nucleotides directly involved in the reaction, and an additional phosphate (dG231) on the 3′ side of dA230 that may activate the nucleophilic water (Figures b and S7). In addition to the residues mentioned above, the QM region for models involving indirect metal–substrate ligation included two additional water molecules coordinated to Mg 2+ to fulfill the octahedral coordination geometry (Figure b), models for direct bidentate metal–substrate ligation included two additional water molecules that hydrogen bonds to D43 or E89 in the QM region (Figure S7a), and models with direct monodentate metal–substrate ligation included an additional water directly coordinated to Mg 2+ to fulfill the octahedral coordination geometry and another water that hydrogen bonds to D43 (Figure S7b).…”

Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila

“…A grid was set to accommodate the active site region with a 0.375 Å span. The torsion and rotatable bonds in the ligands were defined, and the nonpolar hydrogens and partial atomic charges were added to the bonded carbon atoms [23]. The docking was carried out using the AutoDock Vina program [24] to evaluate ligand binding energies over the conformational search space using the Lamarckian genetic algorithm.…”

Rational truncation, mutation, and halogenation of bradykinin neuropeptides as potent ACEII inhibitors by integrating molecular dynamics simulations, quantum mechanics calculations, and in vitro assays

“…A ONIOM-based, two-layered quantum mechanics/ molecular mechanics (QM/MM) was used to minimize the halogen bonds between ACEII with [X]Phecontaining peptides. [31] A protocol modified from previous works [32,33] was employed to partition the complex system, that is, the peptide [X]Phe and the ACEII residue involved in halogen bonding with the [X]Phe were assigned in QM layer, while rest of the complex system were in MM layer. The QM layer was by the density functional theory (DFT) of MPWLYP in conjunction with 6-31G(d) basis set, which has been testified to provide accuracy close to high-level methods in a benchmark study of halogen bonding, [34] while the MM layer was described by universal force field (UFF).…”