On the nature of the transition state in catechol O-methyltransferase. A complementary study based on molecular dynamics and potential energy surface explorations

The way in which enzymes influence the rate of chemical processes is still a question of debate. The protein promotes the catalysis of biochemical processes by lowering the free energy barrier in comparison with the reference uncatalyzed reaction in solution. In this article we are reporting static and dynamic aspects of the enzyme catalysis in a bimolecular reaction, namely a methyl transfer from S-adenosylmethionine to the hydroxylate oxygen of a substituted catechol catalyzed by catechol O-methyltransferase. From QM/MM optimizations, we will first analyze the participation of the environment on the transition vector. The study of molecular dynamics trajectories will allow us to estimate the transmission coefficient from a previously localized transition state as the maximum in the potential of mean force profile. The analysis of the reactive and nonreactive trajectories in the enzyme environment and in solution will also allow studying the geometrical and electronic changes, with special attention to the chemical system movements and the coupling with the environment. The main result, coming from both analyses, is the approximation of the magnesium cation to the nucleophilic and the hydroxyl group of the catecholate as a result of a general movement of the protein, stabilizing in this way the transition state. Consequently, the free energy barrier of the enzyme reaction is dramatically decreased with respect to the reaction in solution.     

On Transition Structures for Hydride Transfer Step in Enzyme Catalysis. A Comparative Study on Models of Glutathione Reductase Derived from Semiempirical, HF, and DFT Methods

As a model of the chemical reactions that take place in the active site of gluthatione reductase, the nature of the molecular mechanism for the hydride transfer step has been characterized by means of accurate quantum chemical characterizations of transition structures. The calculations have been carried out with analytical gradients at AM1 and PM3 semiempirical procedures, ab initio at HF level with 3-21G, 4-31G, 6-31G, and 6-31G basis sets and BP86 and BLYP as density functional methods. The results of this study suggest that the endo relative orientation on the substrate imposed by the active site is optimal in polarizing the C4-Ht bond and situating the system in the neighborhood of the quadratic region of the transition structure associated to the hydride transfer step on potential energy surface. The endo arrangement of the transition structure results in optimal frontier HOMO orbital interaction between NADH and FAD partners. The geometries of the transition structures and the corresponding transition vectors, that contain the fundamental information relating reactive fluctuation patterns, are model independent and weakly dependent on the level of theory used to determine them. A comparison between simple and complex molecular models shows that there is a minimal set of coordinates describing the essentials of hydride transfer step. The analysis of transition vector components suggests that the primary and secondary kinetic isotope effects can be strongly coupled, and this prompted the calculation of deuterium and tritium primary, secondary, and primary and secondary kinetic isotope effects. The results obtained agree well with experimental data and demonstrate this coupling.     

Theoretical modeling of enzyme catalytic power: analysis of "cratic" and electrostatic factors in catechol O-methyltransferase

A comparative theoretical study of a bimolecular reaction in aqueous solution and catalyzed by the enzyme catechol O-methyltransferase (COMT) has been carried out by a combination of two hybrid QM/MM techniques: statistical simulation methods and internal energy minimizations. In contrast to previous studies by other workers, we have located and characterized transition structures for the reaction in the enzyme active site, in water and in a vacuum, and our potential of mean force calculations are based upon reaction coordinates obtained from features of the potential energy surfaces in the condensed media, not from the gas phase. The AM1/CHARMM calculated free energy of activation for the reaction of S-adenosyl methionine (SAM) with catecholate catalyzed by COMT is 15 kcal mol(-1) lower the AM1/TIP3P free-energy barrier for the reaction of the trimethylsulfonium cation with the catecholate anion in water at 300 K, in agreement with previous estimates. The thermodynamically preferred form of the reactants in the uncatalyzed model reaction in water is a solvent-separated ion pair (SSIP). Conversion of the SSIP into a contact ion pair, with a structure resembling that of the Michaelis complex (MC) for the reaction in the COMT active site, is unfavorable by 7 kcal mol(-1), largely due to reorganization of the solvent. We have considered alternative ways to estimate the so-called "cratic" free energy for bringing the reactant species together in the correct orientation for reaction but conclude that direct evaluation of the free energy of association by means of molecular dynamics simulation with a simple standard-state correction is probably the best approach. The latter correction allows for the fact that the size of the unit cell employed with the periodic boundary simulations does not correspond to the standard state concentration of 1 M. Consideration of MC-like species allows a helpful decomposition of the catalytic effect into preorganization and reorganization phases. In the preorganization phase, the substrates are brought together into the MC-like species, either in water or in the enzyme active site. In the reorganization phase, the roles of the enzymic and aqueous environments may be compared directly because reorganization of the substrate is about the same in both cases. Analysis of the electric field along the reaction coordinate demonstrates that in water the TS is destabilized with respect to the MC-like species because the polarity of the solute diminishes and consequently the reaction field is also decreased. In the enzyme, the electric field is mainly a permanent field and consequently there is only a small reorganization of the environment. Therefore, destabilization of the TS is lower than in solution, and the activation barrier is smaller.     

Interface Amorphization of Two-Dimensional Black Phosphorus upon Treatment with Diazonium Salts

Two-dimensional (2D) black phosphorus (BP) represents one of the most appealing 2D materials due to its electronic, optical, and chemical properties. Many strategies have been pursued to face its environmental instability, covalent functionalization being one of the most promising. However, the extremely low functionalization degrees and the limitations in proving the nature of the covalent functionalization still represent challenges in many of these sheet architectures reported to date. Here we shine light on the structural evolution of 2D-BP upon the addition of electrophilic diazonium salts. We demonstrated the absence of covalent functionalization in both the neutral and the reductive routes, observing in the latter case an unexpected interface conversion of BP to red phosphorus (RP), as characterized by Raman, ³¹P-MAS NMR, and X-ray photoelectron spectroscopies (XPS). Furthermore, thermogravimetric analysis coupled to gas chromatography and mass spectrometry (TG-GC-MS), as well as electron paramagnetic resonance (EPR) gave insights into the potential underlying radical mechanism, suggesting a Sandmeyer-like reaction.     

Freezing Capture of Polymorphic Aggregates of Bolaamphiphilic L‐Valine‐Based Molecular Hydrogelators

Nanostructured xerogels have been prepared by the freeze‐drying of hydrogels and aggregates formed by bolaamphiphilic L ‐valine derivatives after aging under different environmental conditions. A wide variety of shapes and sizes has been achieved by a simple methodology. These nanostructures have been studied by SEM and WAXD and a dramatic influence of structural flexibility on the kinetics of aggregation has been observed. Such flexibility and a modulation of the hydrophobic effect have shown a profound influence in the packing of these compounds and revealed a high degree of polymorphism.     

Spatially Resolved Bottom‐Side Fluorination of Graphene by Two‐Dimensional Substrate Patterning

Patterned functionalization can, on the one hand, open the band gap of graphene and, on the other hand, program demanding designs on graphene. The functionalization technique is essential for graphene‐based nanoarchitectures. A new and highly efficient method was applied to obtain patterned functionalization on graphene by mild fluorination with spatially arranged AgF arrays on the structured substrate. Scanning Raman spectroscopy (SRS) and scanning electron microscopy coupled with energy‐dispersive X‐ray spectroscopy (SEM‐EDS) were used to characterize the functionalized materials. For the first time, chemical patterning on the bottom side of graphene was realized. The chemical nature of the patterned functionalization was determined to be the ditopic scenario with fluorine atoms occupying the bottom side and moieties, such as oxygen‐containing groups or hydrogen atoms, binding on the top side, which provides information about the mechanism of the fluorination process. Our strategy can be conceptually extended to pattern other functionalities by using other reactants. Bottom‐side patterned functionalization enables utilization of the top side of a material, thereby opening up the possibilities for applications in graphene‐based devices.     

A prototype calix[4]arene-based receptor for carbohydrate recognition containing peptide and phosphate binding groups

A novel class of macrobicyclic receptors for carbohydrate recognition based on upper rim, peptide-bridged calix[4]arenes has been designed and synthesized. Receptor 12, in which a charged phosphate group cooperates with peptide hydrogen-bonding donor and acceptor groups in the binding process, is the most efficient and selective in the complexation of simple carbohydrate derivatives. The selectivity observed is toward beta-glucoside 13a, which is better bound (DeltaG degrees = 19.6 kJ mol(-)(1)) compared to the corresponding alpha anomer 13b (DeltaG degrees = 17.0 kJ mol(-)(1)) and to the beta-galactoside 13c (DeltaG degrees = 17.7 kJ mol(-)(1)) in CDCl(3). A substantial drop in the stability constant is observed by esterification of the phosphate group in the host 12 or by alkylation of the OH groups in the 2 and 3 positions in the beta-glucoside and beta-galactoside derivatives. On the basis of a careful analysis of the (1)H NMR data available, a binding mode of the beta-octylglucoside 13a to receptor 12 is proposed.     

Preorganization and reorganization as related factors in enzyme catalysis: the chorismate mutase case

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.     

Determination of 3-MCPD by GC-MS/MS with PTV-LV injector used for a survey of Spanish foodstuffs

3-Monochloropropane-1,2-diol (3-MCPD) is the most common chemical contaminant of the group of chloropropanols. It can occur in foods and food ingredients at low levels as a result of processing, migration from packaging materials during storage and domestic cooking. A sensitive method for determination of 3-MCPD in foodstuffs using programmable temperature vaporization (PTV) with large-volume injection (LVI) gas chromatography (GC) with tandem mass spectrometry detection (MS/MS) has been developed and optimized. The optimization of the injection and detection parameters was carried out using statistical experimental design. A Plackett-Burman design was used to estimate the influence of resonance excitation voltage (REV), isolation time (IT), excitation time (ET), ion source temperature (IST), and electron energy (EE) on the analytical response in the ion trap mass spectrometer (ITMS). Only REV was found to have a statically significant effect. On the other hand, a central composite design was used to optimize the settings of injection temperature (T(inlet)), vaporization temperature (T(vap)), vaporization time (t(vap)) and flow (Flow). The optimized method has an instrumental limit of detection (signal-to-noise ratio 3:1) of 0.044 ng mL(-1). From Valencian, Spain, supermarkets 94 samples of foods were surveyed for 3-MCPD. Using the optimized method levels higher than the limit established for soy sauce by the European Union were found in some samples. The estimated daily intake of 3-MCPD throughout the investigated foodstuffs for adults and children was found about 0.005 and 0.01%, respectively, of the established provisional tolerable daily intake.