Peptide Bond Formation Mechanism Catalyzed by Ribosome
In this paper we present a study of the peptide bond formation reaction catalyzed by ribosome. Different mechanistic proposals have been explored by means of Free Energy Perturbation methods within hybrid QM/MM potentials, where the chemical system has been described by the M06-2X functional and the environment by means of the AMBER force field. According to our results, the most favorable mechanism in the ribosome would proceed through an eight-membered ring transition state, involving a proton shuttle mechanism through the hydroxyl group of the sugar and a water molecule. This transition state is similar to that described for the reaction in solution (J. Am. Chem. Soc. 2013, 135, 8708–871…
Heavy enzymes—experimental and computational insights in enzyme dynamics
The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory.
A theoretical study of carbon-carbon bond formation by a Michael-type addition.
A theoretical study of the Michael-type addition of 1,3-dicarbonyl compounds to α,β-unsaturated carbonyl compounds has been performed in the gas phase by means of the AM1 semiempirical method and by density functional theory (DFT) calculations within the B3LYP and M06-2X hybrid functionals. A molecular model has been selected to mimic the role of a base, which is traditionally used as a catalyst in Michael reactions, an acetate moiety to modulate its basicity, and point charges to imitate the stabilization of the negative charge developed in the substrate during the reaction when taking place in enzymatic environments. Results of the study of six different reactions obtained at the three di…
Do zwitterionic species exist in the non-enzymatic peptide bond formation?
The use of proper computational methods and models has allowed answering the controversial question of whether zwitterionic species exist in the mechanism of peptide bond synthesis in aqueous solution. In fact, the different conformations of zwitterionic species open the door to different mechanistic paths.
Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase
In the past decade, L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed since the 1980s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction takes place. In this paper, we show that the chemical step of the LDH-catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single-molecule spectrom…
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, Diels–Alder 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 desig…
Protein Flexibility and Preorganization in the Design of Enzymes. The Kemp Elimination Catalyzed by HG3.17
A recently designed enzyme, HG3.17, obtained by directed evolution, has shown a catalytic activity close to natural enzymes. Hybrid QM/MM molecular dynamics simulations for the Kemp elimination in this new enzyme have provided a deep insight into the origin of its catalytic efficiency. In this case, we have first demonstrated the presence of different conformations with significantly different reactivity. The larger reactivity is related with a better electrostatic preorganization of the environment that creates a more favorable electrostatic potential for the reaction to proceed. In HG3.17, efforts to improve the catalytic properties must be focused in possible mutations increasing the pre…
Unraveling the Reaction Mechanism of Enzymatic C5-Cytosine Methylation of DNA. A Combined Molecular Dynamics and QM/MM Study of Wild Type and Gln119 Variant
M.HhaI is a DNA methyltransferase from Haemophilus hemolyticus that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the C5 position of a cytosine. This enzyme is a paradigmatic model for C5 DNA methyltransferases due to its major homology to mammalian enzymes and to the availability of high-resolution structures of the DNA–enzyme complex. In spite of the number of experimental and theoretical analyses carried out for this system, many mechanistic details remain unraveled. We have used full atomistic classical molecular dynamics simulations to explore the protein–SAM–DNA ternary complex, where the target cytosine base is flipped out into the active site for bot…
Molecular Mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a Twister Ribozyme
Published as part of the special collection of articles derived from the 10th Congress on Electronic Structure: Principles and Applications (ESPA-2016). The catalytic activity of some classes of natural RNA, named as ribozymes, has been discovered just in the past decades. In this paper, the cleavage of the RNA phosphodiester backbone has been studied in aqueous solution and in a twister ribozyme from Oryza sativa. The free energy profiles associated with a baseline substrate-assisted mechanism for the reaction in the enzyme and in solution were computed by means of free energy perturbation methods within hybrid QM/MM potentials, describing the chemical system by the M06-2× functional and t…
Binding isotope effects as a tool for distinguishing hydrophobic and hydrophilic binding sites of HIV-1 RT.
The current treatment for HIV-1 infected patients consists of a cocktail of inhibitors, in an attempt to improve the potency of the drugs by adding the possible effects of each supplied compound. In this contribution, nine different inhibitors of HIV-1 RT, one of the three key proteins responsible for the virus replication, have been selected to develop and test a computational protocol that allows getting a deep insight into the inhibitors’ binding mechanism. The interaction between the inhibitors and the protein have been quantified by computing binding free energies through FEP calculations, while a more detailed characterization of the kind of inhibitor–protein interactions is based on …
Theoretical Studies of the Self Cleavage Pistol Ribozyme Mechanism
AbstractRibozymes are huge complex biological catalysts composed of a combination of RNA and proteins. Nevertheless, there is a reduced number of small ribozymes, the self-cleavage ribozymes, that are formed just by RNA and, apparently, they existed in cells of primitive biological systems. Unveiling the details of these “fossils” enzymes can contribute not only to the understanding of the origins of life but also to the development of new simplified artificial enzymes. A computational study of the reactivity of the pistol ribozyme carried out by means of classical MD simulations and QM/MM hybrid calculations is herein presented to clarify its catalytic mechanism. Analysis of the geometries…
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 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 …
Role of Solvent on Nonenzymatic Peptide Bond Formation Mechanisms and Kinetic Isotope Effects
Based on the hypothesis that similar mechanisms are involved in the peptide bond formation in aqueous solution and in the ribosome, the aminolysis of esters in aqueous solution has been the subject of numerous studies as the reference reaction for the catalyzed process. The mechanisms proposed in the literature have been explored in the present paper by hybrid QM/MM molecular dynamics simulations. The free energy profiles have been computed with the QM region of the system described at semiempirical AM1 level and by DFT within the M06-2X functional. According to the results, the formation of adduct zwitterion species is a preliminary step required for all possible mechanisms. Then, from dif…
Revealing the Origin of the Efficiency of the De Novo Designed Kemp Eliminase HG-3.17 by Comparison with the Former Developed HG-3
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 acc…
Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods
The promiscuous activity of the enzyme o-succinylbenzoate synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both density functional theory and semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylamino acids, with N-succinyl-R-phenylglycine being the best substrate. We investigated the molecular mechanisms for both reactions exploring the potential energy surface. Then, molecular dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues wi…
Extending Limits of Chlorine Kinetic Isotope Effects
Chlorine kinetic isotope effects exceeding semiclassical limits were observed in enzyme-catalyzed reactions, but their source has not been yet identified. Herein we show that unusually large chlorine kinetic isotope effects are associated with reactions in which chlorine is the central atom that is being passed between two heavy atoms. The origin of these large values is the ratio of imaginary frequencies for light-to-heavy species (the so-called temperature-independent factor).
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 Ser1…
Theoretical studies of HIV-1 reverse transcriptase inhibition
Computational methods for accurately calculating the binding affinity of a ligand for a protein play a pivotal role in rational drug design. We herein present a theoretical study of the binding of five different ligands to one of the proteins responsible for the human immunodeficiency virus type 1 (HIV-1) cycle replication; the HIV-1 reverse transcriptase (RT). Two types of approaches are used based on molecular dynamics (MD) simulations within hybrid QM/MM potentials: the alchemical free energy perturbation method, FEP, and the pathway method, in which the ligand is physically pulled away from the binding site, thus rendering a potential of mean force (PMF) for the binding process. Our com…