Clarifying the Catalytic Mechanism of Human Glutamine Synthetase: A QM/MM Study

Glutamine synthetase (GS) is a crucial enzyme responsible for the elimination of both neurotoxic glutamate and toxic ammonium, by combining them into glutamine. Alterations on the GS activity are associated with severe liver and neurodegenerative diseases and its absence or malformation results in death. In this work, the catalytic mechanism of human GS has been investigated with high-level QM/MM calculations, showing a two-phase reaction cycle. During phase 1, GS activates the reactants (NH₄⁺ and glutamate) with extreme efficiency, through NH₄⁺ deprotonation by E305 and glutamate phosphorylation by ATP, in two spontaneous and barrierless reactions. At phase 2, NH₃ attacks the γ-glutamyl phosphate being concomitantly deprotonated by the leaving PO₄^3–, forming the glutamine and HPO₄^2– products. The second phase contains the rate limiting step, with a Δ<i>G</i>^‡ of 19.2 kcal·mol^–1 associated with the nucleophilic substitution of the phosphate by NH₃. The final reaction free energy is −34.5 kcal·mol^–1. Both phases are exergonic, the first by −22.9 kcal·mol^–1 and the second by −11.6 kcal·mol^–1. Direct NH₄⁺ attack is shown to be inefficient; the possible bases that perform the NH₄⁺ deprotonation were systematically investigated. Negative E305 was shown to be the only one possibly responsible for NH₄⁺ deprotonation. Altogether, these results provide a clear atomic level picture of the reaction cycle of GS, consistent with experimental and theoretical studies on GS of this and other organisms, and provide the necessary insights for the development of more specific therapeutic GS inhibitors

Influence of Frozen Residues on the Exploration of the PES of Enzyme Reaction Mechanisms

In this work, we studied one of the very widely used approximations in the prediction of an enzyme reaction mechanism with computational methods, that is, fixing residues outside a given radius surrounding the active site. This avoids the unfolding of truncated models during MD calculations, avoids the expansion of the active site in cluster model calculations (albeit here only specific atoms are frozen), and prevents drifting between local minima when adiabatic mapping with large QM/MM models is used. To test this, we have used the first step of the reaction catalyzed by HIV-1 protease, as the detrimental effects of this approximation are expected to be large here. We calculated the PES with shells of frozen residues of different radii. Models with free regions under a 6.00 Å radius showed signs of being overconstrained. The QM/MM energy barrier for the remaining models was only slightly sensitive to this approximation (average of 0.8 kcal·mol^–1, maximum of 1.6 kcal·mol-1). The influence over the energy of reaction was almost negligible. This widely used approximation seems safe and robust. The resulting error is on average below 1.6 kcal·mol^–1, which is small when compared with others deriving from, for example, the choice of the density functional or semiempirical MO/SCC-DFTB method, the basis set used, or even the lack of sampling or incomplete sampling

QM/MM Study and MD Simulations on the Hypertension Regulator Angiotensin-Converting Enzyme

Human angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that converts angiotensin I to the vasoconstrictor angiotensin II and inactivates the vasodilator bradykinin. This dual ability is vital to blood pressure regulation and management of hypertension. Despite the many enzymatic studies on zinc metallopeptidases, the correct substrate binding mode and catalysis of ACE are still not completely understood. Two buried chloride ions activate the ACE hydrolysis efficiency in a substrate-dependent manner, but the molecular mechanism associated with this activation also remains unclear. In this work, the catalytic mechanism of ACE was studied with atomistic detail, using a hybrid quantum mechanical/molecular mechanical method at the ONIOM­(M06-2X/6-311+G­(d,p):Amber//B3LYP/6-31G­(d):Amber) level. The hydrolytic reaction proceeds via a general acid/base mechanism, in which the first mechanistic step involves the displacement of the zinc-bound water molecule that performs a nucleophilic attack on the scissile carbonyl bond to form an oxyanion that results in a gem-diol intermediate. The second step involves a proton transfer from Glu384 to the peptide nitrogen and a subsequent cleavage of the peptidic bond to yield the products in their neutral forms. The conserved residue Glu384 is ideally aligned and has the ability to slightly rearrange its conformation to act as a highly effective proton shuttle. Our results indicate that the nucleophilic attack is the rate-limiting step of ACE catalysis (barrier of ≈19 kcal/mol), which agrees with the experimental data available. Molecular dynamics simulations on ACE were also performed, and the data reported here provide a structural basis for the chloride-dependent activity of ACE. It was observed that the Cl2 absence allows a conformational rearrangement of the Arg522 side chain, which subsequently makes an electrostatic interaction with the zinc-bound Glu411 and perturbs the metal center polarization role during catalysis

Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase Domain of Human Fatty Acid Synthase

Human fatty acid synthase (hFAS) is a large multienzyme that catalyzes all steps of fatty acid synthesis, which is overexpressed in many cancer cells. Studies have shown that FAS inhibitors exhibit antitumor activity without relevant effects over normal cells. Therefore, the molecular description of active sites in hFAS should stimulate the development of inhibitors as anticancer drug candidates. The malonyl-acetyl transferase (MAT) domain is responsible for loading acetyl-CoA and malonyl-CoA substrates to the acyl-carrier protein (ACP) domain, a carrier for fatty acid reaction intermediates. In this work, we have applied computational QM/MM methods at the DLPNO–CCSD­(T)/CBS:AMBER level of theory to study the MAT reaction mechanism. The results indicate that the initial catalytic stage occurs in two sequential steps: (1) nucleophilic attack on the thioester carbonyl group of the substrate through a concerted pathway that involves a Ser-His dyad and (2) tetrahedral intermediate breakdown and release of the free coenzyme A. The Gibbs activation energies for the first and second steps are 13.0 and 6.4 kcal·mol^–1 and 10.9 and 8.0 kcal·mol^–1, whether the substrate transferred to the MAT domain was acetyl-CoA or malonyl-CoA, respectively. Both Met499 and Leu582 form an oxyanion hole that lodges the negative charge of the substrate carbonyl, lowering the first step energetic barriers for both substrates. The mutation of the Arg606 residue by an alanine severely impairs the malonyl transfer reaction, while leading to a kinetic improvement of the transferase activity for acetyl-CoA, which is in agreement with earlier experimental studies. The results from this work encourage future studies that aim for the full comprehension of the MAT catalytic reaction and for the rational design of novel antineoplastic drugs that target this domain

Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase Domain of Human Fatty Acid Synthase

Human fatty acid synthase (hFAS) is a large multienzyme that catalyzes all steps of fatty acid synthesis, which is overexpressed in many cancer cells. Studies have shown that FAS inhibitors exhibit antitumor activity without relevant effects over normal cells. Therefore, the molecular description of active sites in hFAS should stimulate the development of inhibitors as anticancer drug candidates. The malonyl-acetyl transferase (MAT) domain is responsible for loading acetyl-CoA and malonyl-CoA substrates to the acyl-carrier protein (ACP) domain, a carrier for fatty acid reaction intermediates. In this work, we have applied computational QM/MM methods at the DLPNO–CCSD­(T)/CBS:AMBER level of theory to study the MAT reaction mechanism. The results indicate that the initial catalytic stage occurs in two sequential steps: (1) nucleophilic attack on the thioester carbonyl group of the substrate through a concerted pathway that involves a Ser-His dyad and (2) tetrahedral intermediate breakdown and release of the free coenzyme A. The Gibbs activation energies for the first and second steps are 13.0 and 6.4 kcal·mol^–1 and 10.9 and 8.0 kcal·mol^–1, whether the substrate transferred to the MAT domain was acetyl-CoA or malonyl-CoA, respectively. Both Met499 and Leu582 form an oxyanion hole that lodges the negative charge of the substrate carbonyl, lowering the first step energetic barriers for both substrates. The mutation of the Arg606 residue by an alanine severely impairs the malonyl transfer reaction, while leading to a kinetic improvement of the transferase activity for acetyl-CoA, which is in agreement with earlier experimental studies. The results from this work encourage future studies that aim for the full comprehension of the MAT catalytic reaction and for the rational design of novel antineoplastic drugs that target this domain

Unveiling the Catalytic Mechanism of NADP⁺‑Dependent Isocitrate Dehydrogenase with QM/MM Calculations

We have determined the catalytic mechanism of the human cytosolic homodimeric isocitrate dehydrogenase (hICDH), an enzyme involved in the regulation of tumorogenesis. Our study constitutes the first theoretical attempt to describe the entire catalytic cycle of hICDH. In agreement with earlier experimental proposals, the catalysis was shown to proceed in three steps: (1) NADP⁺ reduction by the isocitrate substrate with the help of the Lys212^B base, (2) β-decarboxylation of the resulting oxalosuccinate, generating an enolate, and (3) protonation of this intermediate by Tyr139^A, giving rise to the α-ketoglutarate product. Our study supports that the β-decarboxylation of oxalosuccinate is the most likely rate-limiting step, with an activation Gibbs free energy of 16.5 kcal mol^–1. The calculated values are in close agreement with the 16–17 kcal mol^–1 range, derived by the application of transition state theory to the reaction rates determined experimentally (11 to 38 s^–1). We emphasize the role of Mg²⁺ and Asp275^A, whose acid/base properties throughout the catalytic cycle were found to lower the barrier to physiologically competent values. Aside from its chemical dual role (as a base, deprotonating Lys212^B, and as an acid, protonating the basic Tyr139^A deprotonated by the enolate intermediate), Asp275^A also establishes hydrogen bonds with Arg132^A and Tyr 139^A that become shorter at critical transition states. These residues were shown to influence both the rate and the efficiency of hICDH. The knowledge drawn in this study provides new insights into future clinical and bioengineering applications of hICDH: namely, in the development of techniques to regulate the growth of glioblastomas and to capture and store carbon dioxide. Moreover, it further extends the comprehension of (1) the hydrogen/charge transfer mechanism that regulates the hydrogenation of NADP⁺ to NADPH, an ubiquitous biochemical reaction, and (2) the role of divalent metals as key structure elements in the family of NAD­(P)⁺-dependent β-decarboxylases

Revisiting Partition in Hydrated Bilayer Systems

We discuss potential-of-mean force convergence issues involving the methods for generating starting structures of umbrella sampling simulations for the study of the water–bilayer permeation of drug-like compounds. We provide a proof of concept of the benefits of potential-energy modification methods, in particular the flooding technique, to enhance the sampling and improve the convergence of the potential-of-mean force. In addition, we also discuss how these potential modifiers could introduce the necessary bias to induce the correct description of other relevant internal degrees of freedom for the compounds, thus providing a more accurate description of the permeation process. Effective methodological approximations are taken into consideration, more specifically the inclusion of atomic polarization, and experimental data are used in the validation of computed values whenever possible. The parametrization of the atomic point charges is always a relevant issue in biomolecular simulations. We discuss the influence of a molecule’s conformation-dependent atomic point charges and their impact on the predicted potential-of-mean force

The Catalytic Mechanism of HIV‑1 Integrase for DNA 3′-End Processing Established by QM/MM Calculations

The development of HIV-1 integrase (INT) inhibitors has been hampered by incomplete structural and mechanistic information. Despite the efforts made to overcome these limitations, only one compound has been approved for clinical use so far. In this work, we have used all experimental information available for INT and similar enzymes, to build a model of the holo-integrase:DNA complex that includes an entire central core domain, a ssDNA GCAGT substrate, and two magnesium ions. Subsequently, we used a large array of computational techniques, which included molecular dynamics, thermodynamic integration, and high-level quantum mechanics/molecular mechanics (QM/MM) calculations to study the possible pathways for the mechanism of 3′ end processing catalyzed by INT. We found that the only viable mechanism to hydrolyze the DNA substrate is a nucleophilic attack of an active site water molecule to the phosphorus atom of the scissile phosphoester bond, with the attacking water being simultaneously deprotonated by an Mg²⁺-bound hydroxide ion. The unstable leaving oxoanion is protonated by an Mg²⁺-bound water molecule within the same elementary reaction step. This reaction has an activation free energy of 15.4 kcal/mol, well within the limits imposed by the experimental turnover. This work significantly improves the fundamental knowledge on the integrase chemistry. It can also contribute to the discovery of leads against HIV-1 infection as it provides, for the first time, accurate transition states structures that can be successfully used as templates for high-throughput screening of new INT inhibitors

Study on the wear and the friction of a metallic pair under lubricated sliding.

Este trabalho trata de um estudo experimental das respostas de desgaste e de atrito encontradas em um sistema deslizante lubrificado. Para tanto, foram realizados ensaios de deslizamento em um equipamento para ensaios de desgaste, adotando-se o dispositivo pino-contra-disco, para ensaios com movimento relativo rotativo contínuo entre as amostras, e o dispositivo pino-contra-placa, para ensaios com movimento relativo alternado, ou oscilatório, entre as amostras. Os materiais metálicos ensaiados foram pinos de aço AISI 52100 e contra-corpos de aço AISI 8640. O óleo lubrificante foi o mineral de base parafínico, IV 100. Foram variadas as condições de aditivação e de contaminação do óleo lubrificante e foram utilizados dois níveis de carregamento mecânico, determinada pela relação velocidade/carga. O desgaste foi estudado por microscopia óptica e eletrônica de varredura, medição da área afetada pelo desgaste, perfilometria das superfícies desgastadas e análise de óleo. O atrito e o potencial de contato foram monitorados ao longo dos ensaios. Os resultados obtidos mostraram que o desgaste dos corpos metálicos foi sensível ao carregamento mecânico, à aditivação e à contaminação do óleo. Diferenças foram notadas nas morfologias superficiais entre os resultados de desgaste dos ensaios rotativos e oscilatórios.This work concerns with experimental study of wear and friction responses of lubricated sliding system. Sliding tests were carried out using pin-on-disk wear testing machine for tests with continuous rotating movement, and the pin-on-plate device, for reciprocating tests between specimens. The metallic test coupons were AISI 52100 steel pins and AISI 8640 steel counter-faces. The used lubricant was paraffinic mineral oil, VI 100. The presence of additives and contamination in the lubricant oil were investigated under two mechanical loading levels, determined by the velocity/load relation. The wear was studied by means of optic and scanning electronic microscopes, perfilometry and dimensional analysis of the worn surfaces and oil analysis. The friction and the contact potential were monitored through out the sliding tests. The results showed that the wear of the metallic materials was susceptible to the mechanical loading, the additive and the contamination existence in the oil. It was observed differences among the wear results of the rotating and the reciprocating tests in terms of surface morphologies

Mechanistic Pathway on Human α‑Glucosidase Maltase-Glucoamylase Unveiled by QM/MM Calculations

The excessive consumption of starch in human diets is associated with highly prevalent chronic metabolic diseases such as type 2 diabetes and obesity. α-Glucosidase enzymes contribute to the digestion of starch into glucose and are thus attractive therapeutic targets for diabetes. Given that the active sites of the various families of α-glucosidases have different sizes and structural features, atomistic descriptions of the catalytic mechanisms of these enzymes can support the development of potent and selective new inhibitors. Maltase-glucoamylase (MGAM), in particular, has a N-terminal catalytic domain (NtMGAM) that has shown high inhibitor selectivity. We provide here the first theoretical study of the human NtMGAM catalytic domain, employing a hybrid QM/MM approach with the ONIOM method to disclose the full atomistic details of the reactions promoted by this domain. We observed that the catalytic activity follows the classical Koshland double-displacement mechanistic pathway that uses general acid and base catalysts. A covalent glycosyl-enzyme intermediate was formed and hydrolyzed in the first and second mechanistic steps, respectively, through oxocarbenium ion-like transition state structures. The overall reaction is of dissociative type. Both transition state geometries differ from those known to occur in other glycosidases. The activation free energy for the glycosylation rate-limiting step agrees with the experimental barrier of 15.8 kcal·mol^–1. Both individual mechanistic steps of the reaction are exoergonic. These structural results may serve as the basis for the design of transition state analogue inhibitors that specifically target the intestinal NtMGAM catalytic domain, thus delaying the production of glucose in diabetic and obese patients