The Hydrolysis of Amides and the Proficiency of Amidohydrolases. The Burden Borne by <i>k</i>_w

The hydrolysis of small amides has garnered major attention due to its relevance to peptide hydrolysis, one of the most fundamental reactions of biology. Both experimental and theoretical research efforts have studied the reaction in different media, and a consensus has been reached regarding the specific acid- and base-catalyzed reaction pathways. Nevertheless, for the water reaction, large discrepancies between theoretical and experimental results are found in the literature. Herein, we report the results of theoretical calculations of formamide and urea hydrolysis at different pH values. Model systems have been built clustering one and two water molecules with the reactive amide. A careful analysis of the reaction pathways at different temperatures has allowed us to accurately reproduce available experimental data and to separate the water reactions from their acid and base counterparts. The relevance of the results in providing an accurate definition of the proficiency of amidohydrolases is discussed

Enzymatic Catalysis of Urea Decomposition: Elimination or Hydrolysis?

We present a high-level quantum chemical study of possible reaction mechanisms associated with the catalytic decomposition of urea by a bioinorganic mimetic of the dinickel active site of urease. We chose the phthalazine−dinickel complexes of Lippard and co-workers, because these mimetics have been shown to hydrolytically degrade urea. High-level quantum chemical methodologies were utilized to identify stable intermediates and transition-state structures along several possible reaction pathways. The computed results were then used to further analyze what may occur in the active site of urease. Valuable information on the latter has been extracted from experimental data, computational approaches, and unpublished molecular dynamics simulations. On the basis of these comparative studies, we propose that both the elimination and hydrolytic pathways may compete for urea decomposition in the active site of urease

Catalyzed Decomposition of Urea. Molecular Dynamics Simulations of the Binding of Urea to Urease^†

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0 Å crystal structure of Benini et al. [(1999) Struct. Folding Des. 7, 205−216] of DAP-inhibited urease (PDB code 3UBP), and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. [(1991) J. Am. Chem. Soc. 113, 8262−8270]. Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time-averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A “protein-assisted proton transfer” mechanism points to Asp221 as crucial for catalysis. An “Asp-mediated proton transfer” involves the transfer of a proton from the bridging OH to an NH2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis is discussed in detail

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Protocol for ANM-restrained-MD simulations and β₂AR conformations.

<p><b>A.</b> Structure of β₂AR. Transmembrane helices 1–7 are labeled by numbers and colored in red, orange, yellow, green, blue, purple, and pink, respectively. Cytoplasmic helix 8 and the short extracellular helix below the binding cavity in extracellular loop 2 are colored in cyan. Ligand- and G-protein binding sites are shown by arrows. The palmitolyl group that is anchored to the membrane from the H8 is also shown in cyan. <b>B.</b> The protocol for generating the ensemble of conformations by ANM-restrained-MD algorithm. <b>C.</b> Ribbon diagrams of β₂AR conformations. Front view (top) and back view (bottom) of β2AR conformers generated by ANM-restrained-MD are shown.</p