Elucidating the Protonation State of the γ-Secretase Catalytic Dyad.

γ-Secretase (GS) is an intramembrane aspartyl protease that participates in the sequential cleavage of C99 to generate different isoforms of the amyloid-ß (Aß) peptides that are associated with the development of Alzheimer's disease. Due to its importance in the proteolytic processing of C99 by GS, we performed pH replica exchange molecular dynamics (pH-REMD) simulations of GS in its apo and substrate-bound forms to sample the protonation states of the catalytic dyad. We found that the catalytic dyad is deprotonated at physiological pH in our apo form, but the presence of the substrate at the active site displaces its monoprotonated state toward physiological pH. Our results show that Asp257 acts as the general base and Asp385 as the general acid during the cleavage mechanism. We identified different amino acids such as Lys265, Arg269, and the PAL motif interacting with the catalytic dyad and promoting changes in its acid-base behavior. Finally, we also found a significant pKa shift of Glu280 related to the internalization of TM6-CT in the GS-apo form. Our study provides critical mechanistic insight into the GS mechanism and the basis for future research on the genesis of Aß peptides and the development of Alzheimer's disease.

Exploring the concerted mechanistic pathway for HIV-1 PR-substrate revealed by umbrella sampling simulation.

HIV-1 protease (HIV-1 PR) is an essential enzyme for the replication process of its virus, and therefore considered an important target for the development of drugs against the acquired immunodeficiency syndrome (AIDS). Our previous study shows that the catalytic mechanism of subtype B/C-SA HIV-1 PR follows a one-step concerted acyclic hydrolysis reaction process using a two-layered ONIOM B3LYP/6-31++G(d,p) method. This present work is aimed at exploring the proposed mechanism of the proteolysis catalyzed by HIV-1 PR and to ensure our proposed mechanism is not an artefact of a single theoretical technique. Hence, we present umbrella sampling method that is suitable for calculating potential mean force (PMF) for non-covalent ligand/substrate-enzyme association/dissociation interactions which provide thermodynamic details for molecular recognition. The free activation energy results were computed in terms of PMF analysis within the hybrid QM(DFTB)/MM approach. The theoretical findings suggest that the proposed mechanism corresponds in principle with experimental data. Given our observations, we suggest that the QM/MM MD method can be used as a reliable computational technique to rationalize lead compounds against specific targets such as the HIV-1 protease.

pH Effects and Cooperativity among Key Titratable Residues for Escherichia coli Glycinamide Ribonucleotide Transformylase.

Human glycinamide ribonucleotide transformylase (GAR Tfase) is a regulatory enzyme in the de novo purine biosynthesis pathway that has been extensively studied as an anticancer target. To some extent, inhibition of GAR Tfase selectively targets cancer cells over normal cells and inhibits purine formation and DNA replication. In this study, we investigated E. coli GAR Tfase, which shares high sequence similarity with the human GAR Tfase, and most functional residues are conserved. Herein, we aim to predict the pH-activity curve through a computational approach. We carried out pH-replica exchange molecular dynamics (pH-REMD) simulations to investigate pH-dependent functions such as structural changes, ligand binding, and catalytic activity. To compute the pH-activity curve, we identified the catalytic residues in specific protonation states, referred to as the catalytic competent protonation states (CCPS), which maintain the structure, keep ligands bound, and facilitate catalysis. Our computed population of CCPS with respect to pH matches well with the experimental pH-activity curve. To compute the microscopic pKa values in the catalytically active conformation, we devised a thermodynamic model that considers the coupling between protonation states of CCPS residues and conformational states. These results allow us to correctly identify the general acid and base catalysts and interpret the pH-activity curve at an atomistic level.

pH-Dependent Conformational Changes Due to Ionizable Residues in a Hydrophobic Protein Interior: The Study of L25K and L125K Variants of SNase.

Ionizable residues in the hydrophobic interior of certain proteins are known to play important roles in life processes like energy transduction and enzyme catalysis. These internal ionizable residues show experimental apparent pKa values having large shifts as compared to their values in solution. In the present work, we study the pH-dependent conformational changes undergone by two variants of staphylococcal nuclease (SNase), L25K and L125K, using pH replica exchange molecular dynamics (pH-REMD) in explicit solvent. Our results show that the observed pKa of Lys25 and Lys125 are significantly different than their pKa in solution. We observed that the internal lysine residues prefer to be water-exposed when protonated at low pH, but they remain buried within the hydrophobic pocket when deprotonated at high pH. Using thermodynamic laws, we estimate the microscopic conformation-specific pKa of the water-exposed and buried conformations of the internal lysine residues and explain their relation to the macroscopic observed pKa values. We present the differences in the microscopic mechanisms that lead to similar experimentally observed apparent pKa of Lys25 and Lys125, and explain the need of thermodynamic models of different complexities to account for our calculations. We see that L25K displays pH-dependent fluctuations throughout the entire ß barrel and the α1 helix. In contrast, pH-independent fluctuations are observed in L125K, primarily limited to the α3 helix. The present computational study offers a detailed atomistic understanding of the determinants of the observed anomalous pKa of internal ionizable residues, bolstering the experimental findings.

Half sandwich complexes of chalcogenated pyridine based bi-(N, S/Se) and terdentate (N, S/Se, N) ligands with (η6-benzene)ruthenium(II): synthesis, structure and catalysis of transfer hydrogenation of ketones and oxidation of alcohols.

The half sandwich complexes [(η(6)-C6H6)Ru(L)Cl][PF6] (1-5) have been synthesized by the reactions of (2-arylchalcogenomethyl)pyridine [L = L1-L3] and bis(2-pyridylmethyl)chalcogenide [L = L4-L5] (chalcogen = S, Se; Ar = Ph/2-pyridyl for S, Ph for Se) with [(η(6)-C6H6)RuCl2]2, at room temperature followed by treatment with NH4PF6. Their HR-MS, (1)H, (13)C{(1)H} and (77)Se{(1)H} NMR spectra have been found characteristic. The single crystal structures of 1-5 have been established by X-ray crystallography. The Ru has pseudo-octahedral half sandwich "piano-stool" geometry. The complexes 1-5 have been found efficient for catalytic oxidation of alcohols with N-methylmorpholine-N-oxide (NMO) and transfer hydrogenation of ketones with 2-propanol (at moderate temperature 80 °C) as TON values are up to 9.9 × 10(3) and 9.8 × 10(3) respectively for the two catalytic reactions. On comparing the required catalyst loading for good conversions and reaction time for the present complexes with those reported in literature for other transfer hydrogenation/oxidation catalysts, it becomes apparent that 1-5 have good promise. The complexes of Se ligands have been found more efficient than their sulphur analogues. The complexes of bidentate ligands are more efficient than those of terdentate, due to difficult bond cleavage in the case of latter. These orders of efficiency are supported by DFT calculations. The calculated bond lengths/angles by DFT are generally consistent with the experimental ones.