75 research outputs found
Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 Mpro by QM/MM computational methods
SARS-CoV-2 Mpro is one of the enzymes essential for the replication process of the virus responsible for the
COVID-19 pandemic. This work is focused on exploring its proteolysis reaction by means of QM/MM
methods. The resulting free energy landscape of the process provides valuable information on the
species appearing along the reaction path and suggests that the mechanism of action of this enzyme,
taking place in four steps, slightly differs from that of other cysteine proteases. Our predictions, which
are in agreement with some recently published experimental data, can be used to guide the design of
COVID-19 antiviral compounds with clinical potential
Molecular Insights into the Substrate-Assisted Mechanism of Viral DNA 3âČ-End Processing in Intasome of Prototype Foamy Virus Integrase from Molecular Dynamic and QM/MM Studies
Integrases participate in two important steps for virus replication such as 3âČ-end processing of viral DNA (vDNA) and nuclear entry of host DNA (hDNA). In this work, insight into the structural changes in intasome of prototype foamy virus integrase (PFV-IN) complexed with vDNA from classical molecular dynamic (MD) simulations are done. Analysis of the results reveal the existence of alternative conformations of the enzyme active site indicating that the 3âČ-end processing reaction can occur according to three different pathways and taking place with the possible participation of aspartate 185 of a neighboring phosphate group or involving an internal phosphate group of the substrate. In this work, one of them, the so-called substrate-assisted mechanism was explored by QM/MM methods. The free energy barriers of 34.4 kcal molâ1 for the first and 35.3 kcal molâ1 for the second step of reaction computed with free energy perturbation (FEP) methods at the M06-2X/AMBER level show that 3âČ-end processing has to proceed via a different mechanism than studied herein. Nevertheless, the obtained results are in good agreement with the experimental observations that the substitution of the key atom for this mechanism, oxygen by sulfur, did not influence the catalysis. Additionally, the obtained mechanism reveals significant similarities to the previously studied substrate-assisted mechanism in twister ribozyme. The possible role of Mg2+ in the active site is discussed
Influence of Dielectric Environment upon Isotope Effects onGlycoside Heterolysis: Computational Evaluation and AtomicHessian Analysis
Isotope effects depend upon the polarity of the bulk medium in which a chemical process occurs. Implicit solvent calculations with molecule-shaped cavities show that the equilibrium isotope effect (EIE) for heterolysis of the glycosidic bonds in 5âČ-methylthioadenosine and in 2-(p-nitrophenoxy)tetrahydropyran, both in water, are very sensitive in the range 2 †Δ †10 to the relative permittivity of the continuum surrounding the oxacarbenium ion. However, different implementations of nominally the same PCM method can lead to opposite trends being predicted for the same molecule. Computational modeling of the influence of the inhomogeneous effective dielectric surrounding a substrate within the protein environment of an enzymic reaction requires an explicit treatment. The EIE (KH/KD) for transfer of cyclopentyl, cyclohexyl, tetrahydrofuranyl and tetrahydropyranyl cations from water to cyclohexane is predicted by B3LYP/6-31+G(d) calculations with implicit solvation and confirmed by B3LYP/6-31+G(d)/OPLS-AA calculations with averaging over many explicit solvation configurations. Atomic Hessian analysis, whereby the full Hessian is reduced to the elements belonging to a single atom at the site of isotopic substitution, reveals a remarkable result for both implicit and explicit solvation: the influence of the solvent environment on these EIEs is essentially captured completely by only a 3 Ă 3 block of the Hessian, although these values must correctly reflect the influence of the whole environment. QM/MM simulation with ensemble averaging has an important role to play in assisting the meaningful interpretation of observed isotope effects for chemical reactions both in solution and catalyzed by enzymes
The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism
The hydride transfer from C6 of tetrahydrofolate to the reaction's exocyclic methyleneâdUMP intermediate is the rate limiting step in thymidylate synthase (TSase) catalysis. This step has been studied by means of QM/MM molecular dynamics simulations to generate the corresponding free energy surfaces. The use of two different initial X-ray structures has allowed exploring different conformational spaces and the existence of chemical paths with not only different reactivities but also different reaction mechanisms. The results confirm that this chemical conversion takes place preferentially via a concerted mechanism where the hydride transfer is conjugated to thiol-elimination from the product. The findings also confirm the labile character of the substrateâenzyme covalent bond established between the C6 of the nucleotide substrate and a conserved cysteine residue. The calculations also reproduce and rationalize a normal H/T 2° kinetic isotope effect measured for that step. From a computational point of view, the results demonstrate that the use of an incomplete number of coordinates to describe the real reaction coordinate can render biased results
Theoretical studies of energetics and binding isotope effects of binding a triazole-based inhibitor to HIV-1 reverse transcriptase
Understanding of protein-ligand interactions is crucial for rational drug design. Binding isotope effects, BIEs, can provide intimate details of specific interactions between individual atoms of an inhibitor and the binding pocket. We have applied multi-scale QM/MM simulations to evaluate binding energetics of a novel triazole-based non-nucleoside inhibitor of HIV-1 reverse transcriptase and to calculate associated BIEs. The binding sites can be distinguished based on the 18O-BIE.This work has been supported by the grants 2011/02/A ST4/00246
(Maestro) from the Polish National Research Center (NCN) and
0478/IP3/2015/73 (Iuventus Plus) from the Polish Ministry of Science and Higher Education
Unlocking a (Pseudo)-Mechanically Interlocked Molecule with a Coronene âShoehornâ
Mechanically interlocked molecules (MIMs) have gained increasing interest during the last decades, not only because of their aesthetic appeal, but also because their unique properties have allowed them to find applications in nanotechnology, catalysis, chemosensing and biomedicine. Herein we describe how a pyrene molecule with four octynyl substituents can be easily encapsulated within the cavity of a tetragold(I) rectangle-like metallobox, by template formation of the metallo-assembly in the presence of the guest. The resulting assembly behaves as a mechanically interlocked molecule (MIM), in which the four long limbs of the guest protrude from the entrances of the metallobox, thus locking the guest inside the cavity of the metallobox. The new assembly resembles a metallo-suit[4]ane, given the number of protruding long limbs and the presence of the metal atoms in the host molecule. However, unlike normal MIMs, this molecule can release the tetra-substituted pyrene guest by the addition of coronene, which can smoothly replace the guest in the cavity of the metallobox. Combined experimental and computational studies allowed the role of the coronene molecule in facilitating the release of the tetrasubstituted pyrene guest to be explained, through a process that we named âshoehorningâ, as the coronene compresses the flexible limbs of the guest so that it can reduce its size to slide in and out the metallobox.Funding for open access charge: CRUE-Universitat Jaume
Theoretical study of primary reaction of Pseudozyma antarctica lipase B as the starting point to understand its promiscuity
Pseudozyma antarctica lipase B (PALB) is a serine hydrolase that catalyzes the hydrolysis of carboxylic acid esters in aqueous medium but it has also shown catalytic activity for a plethora of reactions. This promiscuous activity has found widespread applications. In the present paper, the primary reaction of PALB, its native hydrolytic activity, has been studied using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials. Free energy surfaces, obtained from QM/MM Molecular Dynamics (MD) simulations, show that the reaction takes place by means of a multi-step mechanism where the first step, the activation of the carbonyl group of the substrate and the nucleophilic attack of Ser105 to the carbonyl carbon atom, presents the highest energy transition state. Our results, which are in good agreement with kinetic experimental data, suggest that the origin of the catalytic activity of the enzyme is due to favorable interactions established between the residues of the active site that create an oxyanion hole, Gln106 and Thr40, as well as the Asp187 that is capable of
modulating the pKa of His224 to act as a base or an acid depending on the step of the catalytic process. Our results can be used to rationalize the design of an optimum biocatalyst to accelerate fundamental reactions in organic synthesis based on the protein scaffold of PALB
Computational Study of the Michaelis Complex Formation and the Effect on the Reaction Mechanism of Cruzain Cysteine Protease
Cruzain, a cysteine protease of the papain family, is essential in the development of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease, making it an attractive target for developing new drugs. The present paper is aimed at the study of the catalytic mechanism of the cruzain by first exploring the different protonation states of the active site Cys25 and His159 in the Michaelis complex and the effect on the full catalytic mechanism of this enzyme. The exploration of the equilibrium between these two states has been performed with alchemical free energy perturbation methods with molecular mechanics (MM) force fields and by generating the free energy surfaces in terms of the potential of mean force computed at two levels of theory: AM1d/MM and M06-2X/6-31+G(d,p):AM1d/MM. Alternative mechanisms for the acylation step have been identified on the free energy surfaces and the results suggest the existence of three new reaction mechanisms starting from the peptide binding to the apoenzyme in its neutral Cys25S/His159 dyad state. The mechanism starting with the protonation of the nitrogen atom of the peptide followed by the attack of Cys25Sâ was revealed as the most favorable one, but it can be competitive with its counterpart mechanism initiated in the Cys25Sâ/His159H+ ion pair Michaelis complex. Analysis of energetic and average geometries will allow continuing improvement of our knowledge on this enzyme at the molecular level, which can be crucial to the design of new inhibitors based on the structures of the transition states (transition states analogues) or stable intermediates
Computational strategies for the design of new enzymatic functions
In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, DielsâAlder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on molecular dynamics simulations with Quantum Mechanics/Molecular Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.This work was supported by the Spanish Ministerio de EconomĂa y Competitividad for project CTQ2012-36253-C03, Universitat Jaume I â Spain (project P1·1B2014-26), Generalitat Valenciana â Spain (PROMETEOII/2014/022 and ACOMP/2014/277 projects), Polish National Center for Science (NCN) (grant 2011/02/A/ST4/00246, 2012â2017), the Polish Ministry of Science and Higher Education (âIuventus Plusâ program project no. 0478/IP3/2015/73, 2015-2016) and the USA National Institute of Health (ref. NIH R01 GM065368). Authors acknowledge computational resources from the Servei dâInformĂ tica of Universitat de ValĂšncia on the âTirantâ supercomputer and the Servei dâInformatÌica of Universitat Jaume I
Insights on the Origin of Catalysis on Glycine N-Methyltransferase from Computational Modeling
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 donorâacceptor 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
- âŠ