Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 106
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Phys Chem Lett ; 10(21): 6750-6754, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31609626

RESUMO

Photodynamic therapy is a promising approach to treat a variety of superficial tumors and other diseases. One of its major limitations arises from its dependence on molecular oxygen, which decreases the efficiency of the therapy in hypoxia conditions commonly developed by solid tumors. The present contribution reveals the molecular mechanism of a modified thymine bearing a nitroimidazole substituent, a photosensitizer able to produce highly harmful interstrand cross-links in the DNA double strand after irradiation selectively in absence of oxygen. The mechanism is resolved at a fully atomistic and electronic level relying on quantum mechanics (CASPT2, coupled-cluster, DFT, and TD-DFT methods), classical molecular dynamics, and advanced biased QM/MM simulations, revealing an energy penalty of ∼8 kcal/mol for the anionic nitromidazole release. Our findings indicate that the global interstrand cross-link production is driven by a combination of multiple factors, namely, the reverse energy penalty, the diffusion of the nitroimidazole anion, and the further reactivity of the formed thymine radical. On the basis of these results, we also suggest some possible strategies to improve the efficiency of interstrand cross-link production.


Assuntos
DNA/química , Nitroimidazóis/química , DNA/metabolismo , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Fármacos Fotossensibilizantes/química , Teoria Quântica
2.
PLoS One ; 14(9): e0215793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31483779

RESUMO

Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

3.
ACS Catal ; 9(7): 5902-5911, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31289693

RESUMO

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.

4.
ACS Catal ; 7(5): 3190-3198, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31157122

RESUMO

Hydride transfer is one of the most common reactions catalyzed by enzymatic systems and it has become an object of study due to possible significant quantum tunneling effects. In the present work, we provide a combination of theoretical QM/MM simulations and experimental measurements of the rate constants and kinetic isotopic effects (KIEs) for the hydride transfer reaction catalyzed by morphinone reductase, MR. Quantum mechanical tunneling coefficients, computed in the framework of variational transition-state theory, play a significant role in this reaction, reaching values of 23.8 ± 5.5 for the lightest isotopologue; one of the largest values reported for enzymatic systems. This prediction is supported by the agreement between the theoretically predicted rate constants and the corresponding experimental values. Simulations indicate that the role of protein motions can be satisfactorily described as equilibrium fluctuations along the reaction coordinate, in line with a high degree of preorganization displayed by this enzyme.

5.
Phys Chem Chem Phys ; 21(21): 10908-10913, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31080970

RESUMO

We estimate the time- and temperature-evolution of spin energy levels in a metallopeptide by combining molecular dynamics with crystal field analysis. Fluctuations of tens of cm-1 for spin energy levels at fs times gradually average out at longer times. We confirm that local vibrations are key in spin dynamics.


Assuntos
Metaloproteínas/química , Simulação de Dinâmica Molecular , Termodinâmica , Fenômenos Magnéticos , Fatores de Tempo , Vibração
6.
Chem Sci ; 10(14): 4082-4088, 2019 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-31049190

RESUMO

Enzymatic catalysis is of great importance to the chemical industry. However, we are still scratching the surface of the potential of biocatalysis due to the limited operating range of enzymes in harsh environments or their low recyclability. The role of Metal-Organic Frameworks (MOFs) as active supports to help overcome these limitations, mainly by immobilization and stabilization of enzymes, is rapidly expanding. Here we make use of mild heating and a non-polar medium during incubation to induce the translocation of a small enzyme like protease in the mesoporous MOF MIL-101(Al)-NH2. Our proteolytic tests demonstrate that protease@MIL-101(Al)-NH2 displays higher activity than the free enzyme under all the conditions explored and, more importantly, its usability can be extended to extreme conditions of pH and high temperatures. MOF immobilization is also effective in providing the biocomposite with long-term stability, recyclability and excellent compatibility with competing enzymes. This simple, one-step infiltration strategy might accelerate the discovery of new MOF-enzyme biocatalysts that meet the requirements for biotechnological applications.

7.
Chem Sci ; 10(10): 2882-2892, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30996866

RESUMO

Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli, which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg2+. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.

8.
Chembiochem ; 20(22): 2807-2812, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31016852

RESUMO

An unsolved mystery in biology concerns the link between enzyme catalysis and protein motions. Comparison between isotopically labelled "heavy" dihydrofolate reductases and their natural-abundance counterparts has suggested that the coupling of protein motions to the chemistry of the catalysed reaction is minimised in the case of hydride transfer. In alcohol dehydrogenases, unnatural, bulky substrates that induce additional electrostatic rearrangements of the active site enhance coupled motions. This finding could provide a new route to engineering enzymes with altered substrate specificity, because amino acid residues responsible for dynamic coupling with a given substrate present as hotspots for mutagenesis. Detailed understanding of the biophysics of enzyme catalysis based on insights gained from analysis of "heavy" enzymes might eventually allow routine engineering of enzymes to catalyse reactions of choice.

9.
Eur J Med Chem ; 169: 159-167, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30875506

RESUMO

The metabolic product of caspase-1, IL-1ß, is an important mediator in inflammation and pyroptosis cell death process. Alzheimer's disease, septic shock and rheumatoid arthritis are IL-1ß mediated diseases, making the caspase-1 an interesting target of pharmacological value. Many inhibitors have been developed until now, most of them are peptidomimetic with improved potency. In the present study, all-atom molecular dynamics simulations and the MM/GBSA method were employed to reproduce and interpret the results obtained by in vitro experiments for a series of inhibitors. The analysis shows that the tautomeric state of the catalytic His237 impact significantly the performance of the prediction protocol, providing evidence for a His237 tautomeric state different to the proposed in the putative mechanism. Additionally, analysis of inhibitor-enzyme interactions indicates that the differences in the inhibitory potency of the tested ligands can be explained mainly by the interaction of the inhibitors with the S2-S4 protein region. These results provide guidelines for subsequent studies of caspase-1 catalytic reaction mechanism and for the design of novel inhibitors.


Assuntos
Caspase 1/metabolismo , Desenho de Drogas , Serpinas/farmacologia , Proteínas Virais/farmacologia , Biocatálise , Relação Dose-Resposta a Droga , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Serpinas/síntese química , Serpinas/química , Relação Estrutura-Atividade , Termodinâmica , Proteínas Virais/síntese química , Proteínas Virais/química
10.
Chem Sci ; 9(41): 7902-7911, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30450180

RESUMO

The intrinsic photostability of nucleic acids is intimately related to evolution of life, while its understanding at the molecular and electronic levels remains a challenge for modern science. Among the different decay pathways proposed in the last two decades, the excited-state hydrogen transfer between guanine-cytosine base pairs has been identified as an efficient non-reactive channel to dissipate the excess of energy provided by light absorption. The present work studies the dynamics of such phenomena taking place in a (dG)·(dC) B-DNA homopolymer in water solution using state-of-the-art molecular modelling and simulation methods. A dynamic effect that boosts the photostability of the inter-strand hydrogen atom transfers, inherent to the Watson-Crick base pairing, is unveiled and ascribed to the energy released during the proton transfer step. Our results also reveal a novel mechanism of DNA decay named four proton transfer (FPT), in which two protons of two adjacent G-C base pairs are transferred to form a biradical zwitterionic intermediate. Decay of the latter intermediate to the ground state triggers the transfer of the protons back to the guanine molecules recovering the Watson-Crick structure of the tetramer. This FPT process is activated by the close interaction of a nearby Na+ counterion with the oxygen atoms of the guanine nucleobases and hence represents a photostable channel operative in natural nucleic acids.

11.
J Phys Chem B ; 122(38): 8861-8871, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30157632

RESUMO

Catechol- O-methyltransferase is an enzyme that catalyzes the methylation reaction of dopamine by S-adenosylmethionine, increasing the reaction rate by almost 16 orders of magnitude compared to the reaction in aqueous solution. Here, we combine the recently introduced adaptive string method and the mean reaction force method, in combination with the structural and electronic descriptors to characterize the reaction mechanism. The catalytic effect of the enzyme is addressed by the comparison of the reaction in the human wild-type enzyme, in the less effective Y68A mutant, and in aqueous solution. The influence of these different environments at different stages of the chemical process and the significance of the key collective variables describing the reaction were quantified. Our results show that the native enzyme limits the access of water molecules to the active site, enhancing the interaction between the reactants and providing a more favorable electrostatic environment to assist the SN2 methyl transfer reaction.


Assuntos
Catecol O-Metiltransferase/química , Catálise , Domínio Catalítico , Catecol O-Metiltransferase/genética , Dopamina/química , Humanos , Metilação , Mutação , S-Adenosilmetionina/química , Termodinâmica , Água/química
12.
J Am Chem Soc ; 140(12): 4327-4334, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29460630

RESUMO

The origin of enzyme catalysis remains a question of debate despite much intense study. We report a QM/MM theoretical study of the SN2 methyl transfer reaction catalyzed by a glycine N-methyltransferase (GNMT) and three mutants to test whether recent experimental observations of rate-constant reductions and variations in inverse secondary α-3H kinetic isotope effects (KIEs) should be attributed to changes in the methyl donor-acceptor distance (DAD): Is catalysis due to a compression effect? Semiempirical (AM1) and DFT (M06-2X) methods were used to describe the QM subset of atoms, while OPLS-AA and TIP3P classical force fields were used for the protein and water molecules, respectively. The computed activation free energies and KIEs are in good agreement with experimental data, but the mutations do not meaningfully affect the DAD: Compression cannot explain the experimental variations on KIEs. On the contrary, electrostatic properties in the active site correlate with the catalytic activity of wild type and mutants. The plasticity of the enzyme moderates the effects of the mutations, explaining the rather small degree of variation in KIEs and reactivities.


Assuntos
Glicina N-Metiltransferase/metabolismo , Teoria Quântica , Biocatálise , Glicina N-Metiltransferase/química , Glicina N-Metiltransferase/genética , Cinética , Conformação Molecular , Eletricidade Estática
13.
Angew Chem Int Ed Engl ; 57(12): 3128-3131, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29341402

RESUMO

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.

14.
J Phys Chem A ; 121(51): 9764-9772, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29190105

RESUMO

Here we present a modified version of the on-the-fly string method for the localization of the minimum free energy path in a space of arbitrary collective variables. In the proposed approach the shape of the biasing potential is controlled by only two force constants, defining the width of the potential along the string and orthogonal to it. The force constants and the distribution of the string nodes are optimized during the simulation, improving the convergence. The optimized parameters can be used for umbrella sampling with a path CV along the converged string as the reaction coordinate. We test the new method with three fundamentally different processes: chloride attack to chloromethane in bulk water, alanine dipeptide isomerization, and the enzymatic conversion of isochorismate to piruvate. In each case the same set of parameters resulted in a rapidly converging simulation and a precise estimation of the potential of mean force. Therefore, the default settings can be used for a wide range of processes, making the method essentially parameter free and more user-friendly.

15.
Proc Natl Acad Sci U S A ; 114(47): 12390-12395, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29101125

RESUMO

While being one of the most popular reaction rate theories, the applicability of transition state theory to the study of enzymatic reactions has been often challenged. The complex dynamic nature of the protein environment raised the question about the validity of the nonrecrossing hypothesis, a cornerstone in this theory. We present a computational strategy to quantify the error associated to transition state theory from the number of recrossings observed at the equicommittor, which is the best possible dividing surface. Application of a direct multidimensional transition state optimization to the hydride transfer step in human dihydrofolate reductase shows that both the participation of the protein degrees of freedom in the reaction coordinate and the error associated to the nonrecrossing hypothesis are small. Thus, the use of transition state theory, even with simplified reaction coordinates, provides a good theoretical framework for the study of enzymatic catalysis.


Assuntos
Biocatálise , Simulação de Dinâmica Molecular , Tetra-Hidrofolato Desidrogenase/química , Humanos , Íons/química , Cinética
16.
PLoS One ; 12(10): e0186286, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29045454

RESUMO

Sulfur trafficking in living organisms relies on transpersulfuration reactions consisting in the enzyme-catalyzed transfer of S atoms via activated persulfidic S across protein-protein interfaces. The recent elucidation of the mechanistic basis for transpersulfuration in the CsdA-CsdE model system has paved the way for a better understanding of its role under oxidative stress. Herein we present the crystal structure of the oxidized, inactivated CsdE dimer at 2.4 Å resolution. The structure sheds light into the activation of the Cys61 nucleophile on its way from a solvent-secluded position in free CsdE to a fully extended conformation in the persulfurated CsdA-CsdE complex. Molecular dynamics simulations of available CsdE structures allow to delineate the sequence of conformational changes underwent by CsdE and to pinpoint the key role played by the deprotonation of the Cys61 thiol. The low-energy subunit orientation in the disulfide-bridged CsdE dimer demonstrates the likely physiologic relevance of this oxidative dead-end form of CsdE, suggesting that CsdE could act as a redox sensor in vivo.


Assuntos
Liases de Carbono-Enxofre/química , RNA Helicases DEAD-box/química , Proteínas de Escherichia coli/química , Conformação Proteica , Enxofre/química , Liases de Carbono-Enxofre/genética , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Simulação de Dinâmica Molecular , Estresse Oxidativo/genética , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Enxofre/metabolismo
17.
J Chem Theory Comput ; 13(10): 5089-5096, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-28901132

RESUMO

Oxidation of nucleic acids is ubiquitous in living beings under metabolic impairments and/or exposed to external agents such as radiation, pollutants, or drugs, playing a central role in the development of many diseases mediated by DNA/RNA degeneration. Great efforts have been devoted to unveil the molecular mechanisms behind the OH radical additions to the double bonds of nucleobases; however, the specific role of the biological environment remains relatively unexplored. The present contribution tackles the study of the OH radical addition to uracil from the gas phase to a full RNA macromolecule by means of quantum-chemistry methods combined with molecular dynamics simulations. It is shown that, in addition to the intrinsic reactivity of each position driven by the electronic effects, the presence of bridge water molecules intercalated into the RNA structure favors the addition to the C5 position of uracil in biological conditions. The results also suggest that diffusion of the OH radical does not play a relevant role in the regioselectivity of the reaction, which is mainly controlled at the chemical stage of the addition process.


Assuntos
Radical Hidroxila/química , Teoria Quântica , RNA/química , Uracila/química , Soluções , Estereoisomerismo , Água/química
18.
Chemistry ; 23(31): 7582-7589, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334464

RESUMO

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole.

19.
J Phys Chem B ; 120(50): 12820-12825, 2016 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-28002952

RESUMO

The photoswitching speed of the reversibly switchable fluorescent proteins (RSFPs) from the family of green fluorescent proteins (GFPs) changes upon mutation which is of direct importance for various high-resolution techniques. Dronpa is one of the most used RSFPs. Its point mutants rsFastLime (Dronpa V157G) and rsKame (Dronpa V157L) exhibit a striking difference in their photoswitching speed. Here the QM/MM on-the-fly string method is used in order to explore the details of the thermal isomerization mechanism. The four principal ways in which isomerization may occur have been scrutinized for each of the three proteins. It has been shown that thermal isomerization occurs via a one-bond-flip mechanism in all three proteins, although, in rsKame, where the chromophore is constrained more, the activation free energy difference between hula-twist and one-bond-flip is significantly smaller. Functional mode analysis has been applied to examine the motions of the amino acids during the isomerization. It clearly identifies the importance of Val/Leu 157 as well as the amino acids in the α-helix during the isomerization.


Assuntos
Glicina/química , Proteínas de Fluorescência Verde/química , Leucina/química , Mutação Puntual , Valina/química , Glicina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Isomerismo , Cinética , Leucina/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Temperatura Ambiente , Termodinâmica , Valina/metabolismo
20.
J Phys Chem A ; 120(48): 9636-9646, 2016 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-27933913

RESUMO

In the present work, the reactivity of the tris(benzene-1,2-dithiolato)molybdenum complex ([Mo(bdt)3]) toward water is studied by means of the density functional theory (DFT). DFT calculations were performed using the M06, B3P86, and B3PW91 hybrid functionals for comparison purposes. The M06 method was employed to elucidate the reaction pathway, relative stability of the intermediate products, nature of the Mo-S bond cleavage, and electronic structure of the involved molybdenum species. This functional was also used to study the transference of electrons from the molybdenum center toward the ligands. The reaction pathway confirms that [Mo(bdt)3] undergoes hydrolysis, yielding dihydroxo-bis(benzene-1,2-dithiolato)molybdenum complex ([Mo(OH)2(bdt)2]) and benzenedithiol. The reaction takes place through seven transition structures, one of them involving an aquo seven-coordinate molybdenum intermediate stabilized by a lone pair (LP) LPO→LPMo hyperconjugative interaction. This heptacoordinate species allows understanding of the observed oxygen atom exchange between water and tertiary phosphines mediated by these complexes. Calculations also show that [Mo(C2H4S2)3] and [Mo(OH)2(C2H4S2)2] have d2 and d0 electronic configuration, and hence an electron pair must be transferred during the course of the hydrolysis. The frontier molecular orbital (FMO) analysis concludes that the electron pair is transferred in the rupture of the second Mo-S bond, from the occupied donating Mo dx2-y2 orbital to the unoccupied C2H4(SH)2 S-C σ* ligand orbital. This result is supported by the bond dissociation energy calculations, which demonstrate that the neutral dissociation of the second Mo-S bond is energetically the more favorable.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA