Insights into generalization of the rate-limiting steps of the dehalogenation by LinB and DhaA: A computational approach.

LinB and DhaA are well-known haloalkane dehalogenases (HLDs) capable of converting a plethora of halogenated alkanes, also those considered persistent pollutants. The dehalogenation reaction that these two enzymes catalyze has been studied to determine its rate-limiting step (rls) for the last two decades now. As a result, it has been determined that HLDs can show different rate-limiting steps for individual substrates, and at this point we do not have a basis for any generalization in this matter. Therefore, in this work we aimed at gaining insights into the enzymatic dehalogenation of selected dibromo- and bromochloro-ethanes and propanes by LinB and DhaA using computational approach to determine whether defined structural similarities of the substrates result in a unified mechanism and the same rls. By predicting halogen binding isotope effects (BIEs) as well as computing interaction energy for each HLD-ligand complex the nature of the protein-ligand interactions has been characterized. Furthermore, C and Br kinetic isotope effects (KIEs) as well as the minimum free energy paths (MFEPs) were computed to investigate the chemical reaction for the selected systems. Accuracy of the approach and robustness of the computational predictions were validated by measuring KIEs on the selected reactions. Overall results strongly indicate that any generalization with respect to the enzymatic process involving various ligands in the case of DhaA is impossible, even if the considered ligands are structurally very similar as those analyzed in the present study. Moreover, even small structural differences such as changing of one of the (non-leaving) halogen substituents may lead to significant changes in the enzymatic process and result in a different rls in the case of LinB. It has also been demonstrated that KIEs themselves cannot be used as rls indicators in the reactions catalyzed by the studied HLDs.

Isotope Effects on the Vaporization of Organic Compounds from an Aqueous Solution-Insight from Experiment and Computations.

An isotope fractionation analysis of organic groundwater pollutants can assess the remediation at contaminated sites yet needs to consider physical processes as potentially confounding factors. This study explores the predictability of water-air partitioning isotope effects from experiments and computational predictions for benzene and trimethylamine (both H-bond acceptors) as well as chloroform (H-bond donor). A small, but significant, isotope fractionation of different direction and magnitude was measured with ε = -0.12‰ ± 0.07‰ (benzene), εC = 0.49‰ ± 0.23‰ (triethylamine), and εH = 1.79‰ ± 0.54‰ (chloroform) demonstrating that effects do not correlate with expected hydrogen-bond functionalities. Computations revealed that the overall isotope effect arises from contributions of different nature and extent: a weakening of intramolecular vibrations in the condensed phase plus additional vibrational modes from a complexation with surrounding water molecules. Subtle changes in benzene contrast with a stronger coupling between intra- and intermolecular modes in the chloroform-water system and a very local vibrational response with few atoms involved in a specific mode of triethylamine. An energy decomposition analysis revealed that each system was affected differently by electrostatics and dispersion, where dispersion was dominant for benzene and electrostatics dominated for chloroform and triethylamine. Interestingly, overall stabilization patterns in all studied systems originated from contributions of dispersion rather than other energy terms.

Kinetic Study on the Reactivity of Azanone (HNO) toward Cyclic C-Nucleophiles.

Azanone (HNO) is an elusive electrophilic reactive nitrogen species of growing pharmacological and biological significance. Here, we present a comparative kinetic study of HNO reactivity toward selected cyclic C-nucleophiles under aqueous conditions at pH 7.4. We applied the competition kinetics method, which is based on the use of a fluorescein-derived boronate probe FlBA and two parallel HNO reactions: with the studied scavenger or with O2 (k = 1.8 × 104 M-1s-1). We determined the second-order rate constants of HNO reactions with 13 structurally diverse C-nucleophiles (k = 33-20,000 M-1s-1). The results show that the reactivity of HNO toward C-nucleophiles depends strongly on the structure of the scavenger. The data are supported with quantum mechanical calculations. A comprehensive discussion of the HNO reaction with C-nucleophiles is provided.

Kinetics of Azanone (HNO) Reactions with Thiols: Effect of pH.

HNO (nitroxyl, IUPAC name azanone) is an electrophilic reactive nitrogen species of growing pharmacological and biological significance. Here, we present data on the pH-dependent kinetics of azanone reactions with the low molecular thiols glutathione and N-acetylcysteine, as well as with important serum proteins: bovine serum albumin and human serum albumin. The competition kinetics method used is based on two parallel HNO reactions: with RSH/RS- or with O2. The results provide evidence that the reaction of azanone with the anionic form of thiols (RS-) is favored over reactions with the protonated form (RSH). The data are supported with quantum mechanical calculations. A comprehensive discussion of the HNO reaction with thiolates is provided.

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide.

Hydroethidine (HE) and hydropropidine ([Formula: see text]) are fluorogenic probes used for the detection of the intra- and extracellular superoxide radical anion ([Formula: see text]). In this study, we provide evidence that HE and [Formula: see text] react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with [Formula: see text], leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between [Formula: see text] and the radical cations of the probes. We also synthesized N-methylated analogs of [Formula: see text] and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and [Formula: see text] may be used as a scaffold for the design of a new generation of probes for intra- and extracellular superoxide.

Decomposition of Piloty's acid derivatives - Toward the understanding of factors controlling HNO release.

The recent interest in the clinical applications of Piloty's acid derivatives as HNO donors for the treatment of cardiovascular system dysfunction has led us to the examination of factors controlling HNO release from selected ortho-substituted N-hydroxysulfonamides. Here we present the kinetic and quantum mechanical studies on the mechanism of HNO release from selected ortho-substituted N-hydroxysulfonamides and in vivo examination of the antiaggregatory properties of N-hydroxy-(2-bromobenzene)sulfonamide complex with sodium salt of ß-cyclodextrin sulfobutyl ethers-ethyl ethers as compared with Angeli's salt.

Can Path Integral Molecular Dynamics Make a Good Approximation for Vapor Pressure Isotope Effects Prediction for Organic Solvents? A Comparison to ONIOM QM/MM and QM Cluster Calculation.

Isotopic fractionation of volatile organic compounds (VOCs), which are under strict measures of control because of their potential harm to the environment and humans, has an important ecological aspect, as the isotopic composition of compounds may depend on the conditions in which such compounds are distributed in Nature. Therefore, detailed knowledge on isotopic fractionation, not only experimental but also based on theoretical models, is crucial to follow conditions and pathways within which these contaminants are spread throughout the ecosystems. In this work, we present carbon and, for the first time, bromine vapor pressure isotope effect (VPIE) on the evaporation process from pure-phase systems-dibromomethane and bromobenzene, the representatives of aliphatic and aromatic brominated VOCs. We combine isotope effects measurements with their theoretical prediction using three computational techniques, namely path integral molecular dynamics, QM cluster, and hybrid ONIOM models. While evaporation of both compounds resulted in normal bromine VPIEs, the difference in the direction of carbon isotopic fractionation is observed for the aliphatic and aromatic compounds, where VPIEs are inverse and normal, respectively. Even though theoretical models tested here turned out to be insufficient for quantitative agreement with the experimental values, cluster electronic structure calculations, as well as two-layer ONIOM computations, provided better reproduction of experimental trends.

PIN3 duplication may be partially responsible for TP53 haploinsufficiency.

BACKGROUND: Previously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon. METHODS: 250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed. RESULTS: Haplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures - G-quadruplexes. CONCLUSION: TP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.

WhichCyp: prediction of cytochromes P450 inhibition.

SUMMARY: In this work we present WhichCyp, a tool for prediction of which cytochromes P450 isoforms (among 1A2, 2C9, 2C19, 2D6 and 3A4) a given molecule is likely to inhibit. The models are built from experimental high-throughput data using support vector machines and molecular signatures. AVAILABILITY: The WhichCyp server is freely available for use on the web at http://drug.ku.dk/whichcyp, where the WhichCyp Java program and source code is also available for download.

The contribution of atom accessibility to site of metabolism models for cytochromes P450.

Three different types of atom accessibility descriptors are investigated in relation to site of metabolism predictions. To enable the integration of local accessibility we have constructed 2DSASA, a method for the calculation of the atomic solvent accessible surface area that is independent of 3D coordinates. The method was implemented in the SMARTCyp site of metabolism prediction models and improved the results by up to 4 percentage points for nine cytochrome P450 isoforms. The final models are made available at http://www.farma.ku.dk/smartcyp.

Graphical analysis of pH-dependent properties of proteins predicted using PROPKA.

BACKGROUND: Charge states of ionizable residues in proteins determine their pH-dependent properties through their pKa values. Thus, various theoretical methods to determine ionization constants of residues in biological systems have been developed. One of the more widely used approaches for predicting pKa values in proteins is the PROPKA program, which provides convenient structural rationalization of the predicted pKa values without any additional calculations. RESULTS: The PROPKA Graphical User Interface (GUI) is a new tool for studying the pH-dependent properties of proteins such as charge and stabilization energy. It facilitates a quantitative analysis of pKa values of ionizable residues together with their structural determinants by providing a direct link between the pKa data, predicted by the PROPKA calculations, and the structure via the Visual Molecular Dynamics (VMD) program. The GUI also calculates contributions to the pH-dependent unfolding free energy at a given pH for each ionizable group in the protein. Moreover, the PROPKA-computed pKa values or energy contributions of the ionizable residues in question can be displayed interactively. The PROPKA GUI can also be used for comparing pH-dependent properties of more than one structure at the same time. CONCLUSIONS: The GUI considerably extends the analysis and validation possibilities of the PROPKA approach. The PROPKA GUI can conveniently be used to investigate ionizable groups, and their interactions, of residues with significantly perturbed pKa values or residues that contribute to the stabilization energy the most. Charge-dependent properties can be studied either for a single protein or simultaneously with other homologous structures, which makes it a helpful tool, for instance, in protein design studies or structure-based function predictions. The GUI is implemented as a Tcl/Tk plug-in for VMD, and can be obtained online at http://propka.ki.ku.dk/~luca/wiki/index.php/GUI_Web.

PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions.

In this study, we have revised the rules and parameters for one of the most commonly used empirical pKa predictors, PROPKA, based on better physical description of the desolvation and dielectric response for the protein. We have introduced a new and consistent approach to interpolate the description between the previously distinct classifications into internal and surface residues, which otherwise is found to give rise to an erratic and discontinuous behavior. Since the goal of this study is to lay out the framework and validate the concept, it focuses on Asp and Glu residues where the protein pKa values and structures are assumed to be more reliable. The new and improved implementation is evaluated and discussed; it is found to agree better with experiment than the previous implementation (in parentheses): rmsd = 0.79 (0.91) for Asp and Glu, 0.75 (0.97) for Tyr, 0.65 (0.72) for Lys, and 1.00 (1.37) for His residues. The most significant advance, however, is in reducing the number of outliers and removing unreasonable sensitivity to small structural changes that arise from classifying residues as either internal or surface.

Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of pKa Values.

The new empirical rules for protein pKa predictions implemented in the PROPKA3.0 software package (Olsson et al. J. Chem. Theory Comput.2010, 7, 525-537) have been extended to the prediction of pKa shifts of active site residues and ionizable ligand groups in protein-ligand complexes. We present new algorithms that allow pKa shifts due to inductive (i.e., covalently coupled) intraligand interactions, as well as noncovalently coupled interligand interactions in multiligand complexes, to be included in the prediction. The number of different ligand chemical groups that are automatically recognized has been increased to 18, and the general implementation has been changed so that new functional groups can be added easily by the user, aided by a new and more general protonation scheme. Except for a few cases, the new algorithms in PROPKA3.1 are found to yield results similar to or better than those obtained with PROPKA2.0 (Bas et al. Proteins: Struct., Funct., Bioinf.2008, 73, 765-783). Finally, we present a novel algorithm that identifies noncovalently coupled ionizable groups, where pKa prediction may be especially difficult. This is a general improvement to PROPKA and is applied to proteins with and without ligands.

Mechanistic Analysis of the Base-Catalyzed HF Elimination from 4-Fluoro-4-(4'-nitrophenyl)butane-2-one Based on Liquid-Phase Kinetic Isotope Effects Calculated by Dynamics Modeling with Multidimensional Tunneling.

The primary and secondary deuterium kinetic isotope effects as well as leaving-group fluorine kinetic isotope effects have been calculated for the base-promoted elimination of hydrogen fluoride from 4-fluoro-4-(4'-nitrophenyl)butane-2-one in 75% aqueous methanol solution. The elimination was studied for both formate and imidazole as the catalytic base; and reactant and transition state structures and vibrational frequencies have been calculated by including the base explicitly and by including the solvent by an implicit solvation model that includes both electrostatics by class IV charges and first-solvation-shell effects by atomic surface tensions. We used the M06-L density functional for all calculations. The optimized stationary points, the geometry changes along the solution-phase minimum free energy path, and the solution-phase free energy profile indicate that the elimination reaction occurs concertedly but asynchronously via an E1cb-like transition state. Reaction rates were calculated by the equilibrium solvation path method, using variational transition state theory with multidimensional tunneling. The primary deuterium kinetic isotope effects are calculated to be large: 1.67 and 5.13 for formate and imidazole, respectively. The corresponding C4-secondary deuterium kinetic isotope effects are 1.044 and 1.044, and the leaving group fluorine kinetic isotope effects are respectively 1.020 and 1.015.

Influence of the solvent description on the predicted mechanism of SN2 reactions.

The influence of the implicit solvent model on transition state structures of two S N2 reactions of biochemical importance is presented. In the considered methyl transfer reaction, we show experimentally that the rate constant in blood serum is about 60% slower than in the aqueous solution and that the implicit solvent model with slightly modified parameters for water captures correctly the energetics of this reaction. With the example of the reaction between 4-methyl-1,2,4-triazol-3-thione and ethyl bromoacetate, we show that relative stabilities of the conformationally different transition states depend upon the solvent inclusion strategy.

Enzyme mechanisms from molecular modeling and isotope effects.

The application of kinetic isotope effects and molecular modeling to characterize three enzyme-catalyzed reactions is presented; the mechanism of the chloroacid dehalogenase catalyzed reaction is approached using chlorine kinetic isotope effects and solvent kinetic isotope effects. The pre-steady-state phase of the reaction catalyzed by methylmalonyl-CoA mutase is approached by different QM/MM schemes and the results are validated by comparison with the experimental value of the deuterium kinetic isotope effect. Finally, a procedure for improving QM/MM calculations is illustrated by analysis of the trihydroxynaphthalene reductase-catalyzed reaction.

Analysis of conformer stability for 1,3,8-trihydroxynaphthalene: natural substrate of fungal trihydroxynaphthalene reductase.

1,3,8-Trihydroxynaphthalene is one of the substrates in the fungal melanin biosynthesis pathway. We present theoretical studies on energies of its tautomeric forms and their rotamers. Several theory levels and solvent models have been tested using experimental results obtained in water-acetone mixtures as the reference point. Our results indicate that the best agreement with these data is obtained with density functional theory levels when the continuum solvation model uses the united atom topological cavity. We also noticed a fairly good performance of the semiempirical AM1 method that makes it a promising alternative for studies of large systems.

A new interpretation of chlorine leaving group kinetic isotope effects; a theoretical approach.

The chlorine leaving group kinetic isotope effects (KIEs) for the S(N)2 reactions between methyl chloride and a wide range of anionic, neutral, and radical anion nucleophiles were calculated in the gas phase and, in several cases, using a continuum solvent model. In contrast to the expected linear dependence of the chlorine KIEs on the C(alpha)-Cl bond order in the transition state, the KIEs fell in a very small range (1.0056-1.0091), even though the C(alpha)-Cl transition state bond orders varied widely from approximately 0.32 to 0.78, a range from reactant-like to very product-like. This renders chlorine KIEs, and possibly other leaving-group KIEs, less useful for studies of reaction mechanisms than commonly assumed. A partial explanation for this unexpected relationship between the C(alpha)-Cl transition state bond order and the magnitude of the chlorine KIE is presented.

Validation of semiempirical methods for modeling of corrinoid systems.

Several semiempirical methods (MNDO-d, PM3tm, PM3-d, PM5, PM6, and AM1-d) have been tested against experimental data and density functional theory (DFT) results in search for the best methods that can be used for quantum-mechanical-molecular mechanics (QM/MM) modeling of corrinoid systems of vitamin B(12) co-factor. It has been found that the PM6 parametrization in its present form gives results closest to hybrid DFT calculations that are most widely used thus far. In comparison with pure DFT and experimental data the best agreement is obtained for PM3tm parametrization, while PM6 yields slightly worse results. AM1-d yields bad geometry of the corrin moiety. The worst performance was observed for MNDO-d, which has severe problem with position and orientation of the alpha-ligands.

Calculations of substituent and solvent effects on the kinetic isotope effects of Menshutkin reactions.

Nitrogen, deuterium, halogen, and carbon kinetic isotope effects have been modeled for the Menshutkin reaction between methyl halides and substituted N,N-dimethylaniline at the HF/6-31G(d) level of theory augmented by the C-PCM continuum solvent model for several solvents. Systematic changes in geometries of the transition states and Gibbs free energies of activation have been found with phenyl ring substituents, solvent, and the leaving group. Kinetic isotope effects also change systematically; however, these changes are predicted to be small, inside the usual precision of the experimental measurements. On the contrary, no correlation has been found between the kinetic isotope effects and the Hammett constants for para substituents. Thus opposite to previous assumptions, our results indicate that kinetic isotope effects on the Menshutkin reaction cannot be used to predict the position of the transition state on the reaction coordinate.