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The two divalent metal ions present in the exo site of DNA pol I exert an electrostatic influence during the phosphodiester bond hydrolysis. The first metal ion facilitates electrostatic stabilization to the OH- nucleophile and the second metal ion stabilizes the negative charge on the phosphate. The second metal ion is not involved in the strain mechanism. Sites that bound to the metal ion in a rigid environment decreases the capacity of the metal toward the phosphate center during the nucleophilic attack.

The linear free energy relationships for a series of six adiabatic reactions in solutions were analyzed by computer simulation methods. The simulations evaluated the hydride transfer reactions within a class of NAD+ analogues. Quantum mechanical nuclear corrections of the activation energies of the solute and solvent were analyzed by the quantized classical path method. The solute and solvent energies obtained from the microscopic parameter are different than those obtained by the phenomenological approach.

A theoretical study of the reaction of tyrosyl-tRNA-synthetase (TTS) to examine the basis of linear free energy relationships (LFER) in enzymatic reactions proves the efficiency of pure valence band (VB) resonance structures in analyzing LFER. This VB representation uses Marcus-type concepts to determine LFER in proteins and to analyze reaction mechanisms. This approach helps determine the value of beta in the equation relating changes in activation free energies due to perturbations and free energy difference between reactants and products.

Empirical valence bond (EVB) method facilitates the simulation of enzyme interactions, and is applicable to charge separation and translocation reactions, in addition to reactions incorporating resonance structure. EVB method's forte is its potential simulation of diabatic free-energy surfaces of the essential resonance structures, which enables the substantial microscopic validation of linear free-energy relationships (LFERs). LFERs are applicable to the enzyme-active sets, subject to the condition that they correspond to the intrinsic resonance structural energetics addition to the energetics of reactants and products.

Experimental 19F chemical shifts of the fluorine nuclear magnetic resonance spectrum of the galactose binding protein from Escherichia coli are controlled by weak or long-range electrical interactions. These can be derived by using the responses of the shielding tensor elements to the uniform field components and the non uniform or gradient terms, along with the mean values of the fields (Ex) and field gradients (Vii) obtained from molecular dynamics (MD) trajectories. The 19F NMR shielding of the pentapeptide Gly-Gly(5-F) Trp-Gly-Gly in water is close to that of the denatured protein. Van der Waals interactions are not significant for fluorine shielding non equivalence in proteins, as the experimental outcomes can be acquired by using the computed field and field gradient terms. The ability to predict protein NMR shielding patterns and ranges may be useful for structural analysis and for verifying the varying techniques of deriving protein electrostatics.

The computer simulations of the CO2/HCO3-interconversion step in human carbonic anhydrase are repoprted. The reaction potential energy surface and reaction free energy profiles are constructed using the empirical valence method and a combination of the free energy perturbation technique and molecular dynamics, respectively. The simulated results are found to be in good agreement with experimental estimates of the reaction energetics.

The general base (GB61) hydrolysis mechanism proposes that Gln61 is the general base that facilitates the nucleophilic attack on the gamma-phosphate by activating a water molecule. The role of Gln 61 in ras p21 was examined by the empirical valence bond (EVB) approach which simulates the correlations of structure and energetics of the system by a computer. Free energy perturbation calculations and well-defined electrostatic considerations were used to verify EVB results. The findings indicate that Gln 61 is not likely the general base in the hydrolysis reaction. Alternatively, gamma-phosphate might act as a general base while Gln 61 only stabilizes the OH- nucleophile.

Understanding the activation mechanism of the μ-opioid receptor (μ-OR) and its selective coupling to the inhibitory G protein (Gi) is vital for pharmaceutical research aimed at finding treatments for the opioid overdose crisis. Many attempts have been made to understand the mechanism of the μ-OR activation, following the elucidation of new crystal structures such as the antagonist- and agonist-bound μ-OR. However, the focus has not been placed on the underlying energetics and specificity of the activation process. An energy-based picture would not only help to explain this coupling but also help to explore why other possible options are not common. For example, one would like to understand why μ-OR is more selective to Gi than a stimulatory G protein (Gs). Our study used homology modeling and a coarse-grained model to generate all of the possible “end states” of the thermodynamic cycle of the activation of μ-OR. The end points were further used to generate reasonable intermediate structures of the receptor and the Gi to calculate two-dimensional free energy landscapes. The results of the landscape calculations helped to propose a plausible sequence of conformational changes in the μ-OR and Gi system and for exploring the path that leads to its activation. Furthermore, in silico alanine scanning calculations of the last 21 residues of the C terminals of Gi and Gs were performed to shed light on the selective binding of Gi to μ-OR. Overall, the present work appears to demonstrate the potential of multiscale modeling in exploring the action of G protein-coupled receptors. [ABSTRACT FROM AUTHOR]

Understanding the reaction mechanism of CRISPR‐associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo‐ and substrate‐bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg2+. Trying this mechanism with the cryo‐EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg2+ and assume that the cryo‐EM structure by itself is a suitable representation of a catalytic‐ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell. [ABSTRACT FROM AUTHOR]

G‐protein‐coupled receptors (GPCRs) are among the most important receptors in human physiology and pathology. They serve as master regulators of numerous key processes and are involved in as well as cause debilitating diseases. Consequently, GPCRs are among the most attractive targets for drug design and pharmaceutical interventions (>30% of drugs on the market). The glucagon‐like peptide 1 (GLP‐1) hormone receptor GLP1R is closely involved in insulin secretion by pancreatic β‐cells and constitutes a major druggable target for the development of anti‐diabetes and obesity agents. GLP1R structure was recently solved, with ligands, allosteric modulators and as part of a complex with its cognate G protein. However, the translation of this structural data into structure/function understanding remains limited. The current study functionally characterizes GLP1R with special emphasis on ligand and cellular partner binding interactions and presents a free‐energy landscape as well as a functional model of the activation cycle of GLP1R. Our results should facilitate a deeper understanding of the molecular mechanism underlying GLP1R activation, forming a basis for improved development of targeted therapeutics for diabetes and related disorders. [ABSTRACT FROM AUTHOR]

A comparative study of several different simulation approaches is conducted to explain their applicability for an effective computer-aided design of effective enzymes. The empirical valence bond (EVB) model is shown to be an extremely significant tool for the final stages of the designing process.

The free energy surfaces for both hydroxide and water attack on the dineopentyl phosphate anion and for water attack on the neutral diester were mapped to examine the hydrolysis of dineopentyl phosphate over the entire pH range. The data obtained revealed that the substrate-as-base mechanism is viable for phosphate ester hydrolysis and might have significant implications for the studies of phosphate ester hydrolysis by enzymes.

Wave function propagation approaches are used to simulate isotope effects in enzymes with focus on the large H/D kinetic isotope effect of soybean lipoxygenase-1. The H/D kinetic isotope effect is evaluated as the ratio of the rate constants for hydrogen and deuterium transfer.

Several quantum mechanical/ molecular mechanics (QM/MM) free energy calculations are conducted to study the solvent effects on surroundings by an updated mean charge distribution. The approach accurately evaluates the solvation free energies and can be hence used for several other cases.

An approach to generate ab initio potential energy surfaces for the hydrolysis of phosphate diesters in solution with a variety of leaving groups is described. The findings based on the mechanism for the hydrolysis of phosphate diesters in solution can be applied to study other mechanism of a range of phosphodiesterases such as alkaline phosphatase and DNA polymerases.

The frozen density functional (FDFT) approach is used for generating diabatic surfaces, which are used for generating a mapping potential that has taken the system from the reactant to the product state. The results have shown that the present implementation of the FDFT approach has provided a very good approach for evaluating ab initio quantum mechanical/ molecular mechanics (QM(ai)/MM ) free energy surfaces.

An analysis of DNA polymerase [beta]-catalyzed nucleotidyl transfer mechanism that provides insight into the structural differences of transition states for correct and mispair incorporation of DNA polymerase [beta] system is reported.

The enzyme dehydrofolate reductase and its mutants depend upon the changes in the reorganization energy for their catalytic activity. The mutants show an increase in the distance between the donor and acceptor reducing available free energy for reorganization of polar groups.

The dGTP analogues replacing the [beta],[gamma]-bridging O with C[H.sub.2], CHF, C[F.sub.2], or C[Cl.sub.2] was used to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) [beta]. The location of the breaks relative to p[K.sub.a]s of C[F.sub.2], O, and the sterically bulky C[Cl.sub.2]-bridging compounds suggests that the biphasic nature of the Bronsted correlations is due to a modification-induced change in the mechanism by stabilization of leaving-group elimination.

The nature of the hydrolysis of phosphate monoester dianions in solutions and in proteins is explored by systematic calculations of the potential surfaces of the reactions of a series of phosphate monoesters with different leaving groups. The results have indicated that the nature of the potential surfaces for the phosphoryl transfer reactions in solution and proteins is complicated and cannot be determined in a conclusive way without the use of theoretical studies.

An investigation was carried out to explore the long-standing question of the validity of the empirical valence bond's (EVB's) key assumption that the off diagonal mixing terms are transferable between different environments. The findings reveal that at least for the test case of [S.sub.N]2 reactions, the off diagonal element does not change significantly upon moving from the gas phase to solutions and thus the EVB assumption is valid and extremely useful.

An explanation is provided on the origin of the catalytic power of enzymes by clarifying the confusions regarding the reference state by introducing a catalytic scale that does not include the well understood effect of having different mechanisms in the enzyme and in solution as well as the affect of the binding of the reactant state. A demonstration is presented that the effect of the enzyme environment can be much larger than the estimated 15 kcal/mol

Charge induced structural changes are studied by a new approach by observing the [pK.sub.a] of the Val66Glu mutant of staphylococcal nuclease. It is shown that standard free energy perturbation (FEP) approaches give extremely disappointing results while the new approach gives realistic results and the reduced [pK.sub.a] reflects the protein structural change and water penetration process and the barrier for partial unfolding cannot be surmounted by the regular FEP approach.

The origin of the catalytic power of the ribosome is addressed by using computer simulation approaches and comparing the energetics of the peptide bond formation in the ribosome and in water. Findings reveal that a significant part of the observed activation entropy of the reference solution reaction is due to solvation entropy, and that the proximity effect is smaller than previously thought.

Reliable studies of enzymatic reactions by combined quantum mechanics/molecular mechanics (QM/MM) approaches, with an ab initio description of the quantum region, presents a major challenge to computational chemists. The hypothetical reaction of a metaphosphate ion with water in the Ras. GAP complex was chosen, to examine the problems with energy minimization QM/MM approaches.