A computational study of the phosphoryl transfer reaction between ATP and Dha in Aqueous Solution

Phosphoryl transfer reactions are ubiquitous in biology, being involved in processes ranging from energy and signal transduction to the replication genetic material. Dihydroxyacetone phosphate (Dha-P), an intermediate of the synthesis of pyruvate and a very important building block in nature, can be generated by converting free dihydroxyacetone (Dha) through the action of the dihydroxyacetone kinase enzyme. In this paper the reference uncatalyzed reaction in solution has been studied in order to define the foundations of the chemical reaction and to determine the most adequate computational method to describe this electronically complex reaction. In particular, the phosphorylation reaction mechanism between adenosine triphosphate (ATP) and Dha in aqueous solution has been studied by means of quantum mechanics/molecular mechanics (QM/MM) Molecular Dynamics (MD) simulations with the QM subset of atoms described with semi-empirical and DFT methods. The results appear to be strongly dependent on the level of calculation, which will have to be taken into account for future studies of the reaction catalyzed by enzymes. In particular, PM3/MM renders lower free energy barriers and a less endergonic process than AM1d/MM and PM6/MM methods. Nevertheless, the concerted pathway was not located with the former combination of potentials.Spanish Ministerio de Economia y Competitividad. CTQ2012-36253-C03-01 Generalitat Valenciana. II/2014/022 Universitat Jaume I. P1 1B2013-5

Why Are Some Enzymes Dimers? Flexibility and Catalysis in Thermotoga maritima Dihydrofolate Reductase

Dihydrofolate reductase from Thermotoga maritima (TmDFHFR) is a dimeric thermophilic enzyme that catalyzes the hydride transfer from the cofactor NADPH to dihydrofolate less efficiently than other DHFR enzymes, such as the mesophilic analogue Escherichia coli DHFR (EcDHFR). Using QM/MM potentials, we show that the reduced catalytic efficiency of TmDHFR is most likely due to differences in the amino acid sequence that stabilize the M20 loop in an open conformation, which prevents the formation of some interactions in the transition state and increases the number of water molecules in the active site. However, dimerization provides two advantages to the thermophilic enzyme: it protects its structure against denaturation by reducing thermal fluctuations and it provides a less negative activation entropy, toning down the increase of the activation free energy with temperature. Our molecular picture is confirmed by the analysis of the temperature dependence of enzyme kinetic isotope effects in different DHFR enzymes

Temperature dependence of dynamic, tunnelling and kinetic isotope effects in formate dehydrogenase

The origin of the catalytic power of enzymes has been a question of debate for a long time. In this regard, the possible contribution of protein dynamics in enzymatic catalysis has become one of the most controversial topics. In the present work, the hydride transfer step in the formate dehydrogenase (FDH EC 1.2.1.2) enzyme is studied by means of molecular dynamic (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials in order to explore any correlation between dynamics, tunnelling effects and the rate constant. The temperature dependence of the kinetic isotope effects (KIEs), which is one of the few tests that can be studied by experiments and simulations to shed light on this debate, has been computed and the results have been compared with previous experimental data. The classical mechanical free energy barrier and the number of recrossing trajectories seem to be temperature-independent while the quantum vibrational corrections and the tunnelling effects are slightly temperature-dependent over the interval of 5–45 °C. The computed primary KIEs are in very good agreement with previous experimental data, being almost temperature-independent within the standard deviations. The modest dependence on the temperature is due to just the quantum vibrational correction contribution. These results, together with the analysis of the evolution of the collective variables such as the electrostatic potential or the electric field created by the protein on the key atoms involved in the reaction, confirm that while the protein is well preorganised, some changes take place along the reaction that favour the hydride transfer and the product release. Coordinates defining these movements are, in fact, part of the real reaction coordinate

Unveiling the Mechanistic Singularities of Caspases: A Computational Analysis of the Reaction Mechanism in Human Caspase-1

Caspases are cysteine proteases in charge of breaking a peptide bond next to an aspartate residue. Caspases constitute an important family of enzymes involved in cell death and inflammatory processes. A plethora of diseases, including neurological and metabolic diseases and cancer, are associated with the poor regulation of caspase-mediated cell death and inflammation. Human caspase-1 in particular carries out the transformation of the pro-inflammatory cytokine pro-interleukin-1β into its active form, a key process in the inflammatory response and then in many diseases, such as Alzheimer’s disease. Despite its importance, the reaction mechanism of caspases has remained elusive. The standard mechanistic proposal valid for other cysteine proteases and that involves the formation of an ion pair in the catalytic dyad is not supported by experimental evidence. Using a combination of classical and hybrid DFT/MM simulations, we propose a reaction mechanism for the human caspase-1 that explains experimental observations, including mutagenesis, kinetic, and structural data. In our mechanistic proposal, the catalytic cysteine, Cys285, is activated after a proton transfer to the amide group of the scissile peptide bond, a process facilitated by hydrogen-bond interactions with Ser339 and His237. The catalytic histidine does not directly participate in any proton transfer during the reaction. After formation of the acylenzyme intermediate, the deacylation step takes place through the activation of a water molecule by the terminal amino group of the peptide fragment formed during the acylation step. The overall activation free energy obtained from our DFT/MM simulations is in excellent agreement with the value derived from the experimental rate constant, 18.7 vs 17.9 kcal·mol–1, respectively. Simulations of the H237A mutant support our conclusions and agree with the reported reduced activity observed for this caspase-1 variant. We propose that this mechanism can explain the reactivity of all cysteine proteases belonging to the CD clan and that differences with respect to other clans could be related to the larger preference showed by enzymes of the CD clan for charged residues at position P1. This mechanism would avoid the free energy penalty associated with the formation of an ion pair. Finally, our structural description of the reaction process can be useful to assist in the design of inhibitors of caspase-1, a target in the treatment of several human diseases.The authors thank financial support from grant PID2021-123332OB funded by MCIN/AEI/10.13039/501100011033/ and by “ERDF A way of making Europe” and also from grant PROMETEO CIPROM/2021/079 of Generalitat Valenciana. K.Z. acknowledges a Maria Zambrano fellowship by Ministerio de Universidades (Spain). Computational resources were provided by Barcelona Supercomputing Center and Servei d’Informàtica, Universitat de València (Tirant supercomputer)

Loss of Hyperconjugative Effects Drives Hydride Transfer during Dihydrofolate Reductase Catalysis

Hydride transfer is widespread in nature and has an essential role in applied research. However, the mechanisms of how this transformation occurs in living organisms remain a matter of vigorous debate. Here, we examined dihydrofolate reductase (DHFR), an enzyme that catalyzes hydride from C4′ of NADPH to C6 of 7,8-dihydrofolate (H2F). Despite many investigations of the mechanism of this reaction, the contribution of polarization of the π-bond of H2F in driving hydride transfer remains unclear. H2F was stereospecifically labeled with deuterium β to the reacting center, and β-deuterium kinetic isotope effects were measured. Our experimental results combined with analysis derived from QM/MM simulations reveal that hydride transfer is triggered by polarization at the C6 of H2F. The σ Cβ–H bonds contribute to the buildup of the cationic character during the chemical transformation, and hyperconjugation influences the formation of the transition state. Our findings provide key insights into the hydride transfer mechanism of the DHFR-catalyzed reaction, which is a target for antiproliferative drugs and a paradigmatic model in mechanistic enzymology

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates

QM/MM Study of L-Lactate Oxidation by Flavocytochrome b2

In this work, we have performed molecular dynamics simulations using a hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) scheme to study the mechanism of L-lactate oxidation by flavocytochrome b2 (Fcb2). Our results obtained at the QM(AM1)/MM level have been improved by single-point corrections using density functional theory (DFT) methods. Free energy surfaces have been calculated in the framework of the hydride transfer hypothesis. This mechanism involves the transfer of the lactate hydroxyl proton to H373 while the substrate αH atom is transferred as a hydride to the flavin mononucleotide (FMN) prosthetic group anchored in the active site. Four different systems have been modeled: wild-type enzyme considering R289 in a distal or a proximal conformation observed in crystal structures and the D282N and Y254L variants (with R289 in a distal position). Simulation results highlight the influence of the environment on the catalytic mechanism by describing a step-wise process in the WT enzyme with R289 in a distal position and a concerted mechanism for the other systems. In the step-wise mechanism, the hydride transfer to flavin can occur only after a proton transfer from substrate to H373. Modifications of the electrostatic field around L-lactate or H373 disfavor the highly charged complex resulting from this proton transfer. Simulations of the Y254L variant also reveal some effect of steric changes

III Jornadas de Jóvenes Investigadores en Arqueología: Libro de Actas

Libro de Actas de las III Jornadas de Jóvenes Investigadores en Arqueologí

Congreso Internacional de Responsabilidad Social Apuestas para el desarrollo regional.

Congreso Internacional de Responsabilidad Social: apuestas para el desarrollo regional [Edición 1 / Nov. 6 - 7: 2019 Bogotá D.C.]El Congreso Internacional de Responsabilidad Social “Apuestas para el Desarrollo Regional”, se llevó a cabo los días 6 y 7 de noviembre de 2019 en la ciudad de Bogotá D.C. como un evento académico e investigativo liderado por la Corporación Universitaria Minuto de Dios -UNIMINUTO – Rectoría Cundinamarca cuya pretensión fue el fomento de nuevos paradigmas, la divulgación de conocimiento renovado en torno a la Responsabilidad Social; finalidad adoptada institucionalmente como postura ética y política que impacta la docencia, la investigación y la proyección social, y cuyo propósito central es la promoción de una “sensibilización consciente y crítica ante las situaciones problemáticas, tanto de las comunidades como del país, al igual que la adquisición de unas competencias orientadas a la promoción y al compromiso con el desarrollo humano y social integral”. (UNIMINUTO, 2014). Dicha postura, de conciencia crítica y sensibilización social, sumada a la experiencia adquirida mediante el trabajo articulado con otras instituciones de índole académico y de forma directa con las comunidades, permitió establecer como objetivo central del evento la reflexión de los diferentes grupos de interés, la gestión de sus impactos como elementos puntuales que contribuyeron en la audiencia a la toma de conciencia frente al papel que se debe asumir a favor de la responsabilidad social como aporte seguro al desarrollo regional y a su vez al fortalecimiento de los Objetivos de Desarrollo Sostenible