Theoretical Comparison of <i>p</i>-Nitrophenyl Phosphate and Sulfate Hydrolysis in Aqueous Solution: Implications for Enzyme-Catalyzed Sulfuryl Transfer

Both phosphoryl and sulfuryl transfers are ubiquitous in biology, being involved in a wide range of processes, ranging from cell division to apoptosis. Additionally, it is becoming increasingly clear that enzymes that can catalyze phosphoryl transfer can often cross-catalyze sulfuryl transfer (and vice versa). However, while there have been extensive experimental and theoretical studies performed on phosphoryl transfer, the body of available research on sulfuryl transfer is comparatively much smaller. The present work presents a direct theoretical comparison of <i>p</i>-nitrophenyl phosphate and sulfate monoester hydrolysis, both of which are considered prototype systems for probing phosphoryl and sulfuryl transfer, respectively. Specifically, free energy surfaces have been generated using density functional theory, by initial geometry optimization in PCM using the 6-31+G* basis set and the B3LYP density functional, followed by single-point calculations using the larger 6-311+G** basis set and the COSMO continuum model. The resulting surfaces have been then used to identify the relevant transition states, either by further unconstrained geometry optimization or from the surface itself where possible. Additionally, configurational entropies were evaluated using a combination of the quasiharmonic approximation and the restraint release approach and added to the calculated activation barriers as a correction. Finally, the overall activation entropy was estimated by approximating the solvent contribution to the total activation entropy using the Langevin dipoles solvation model. We have reproduced both the experimentally observed activation barriers and the observed trend in the activation entropies with reasonable accuracy, as well as providing a comparison of calculated and observed ¹⁵N and ¹⁸O kinetic isotope effects. We demonstrate that, counterintuitively, the hydrolysis of the <i>p</i>-nitrophenyl <i>sulfate</i> proceeds through a more expansive pathway than its phosphate analogue. Additionally, we show that the solvation effects upon moving from the ground state to the transition state are quite different for both reactions, suggesting that the enzymes that catalyze these reactions would need active sites with quite different electrostatic preorganization for the efficient catalysis of either reaction (despite which many enzymes can catalyze both phosphoryl and sulfuryl transfer). We believe that such a comparative study is an important foundation for understanding the molecular basis for phosphate–sulfate cross-promiscuity within members of the alkaline phosphatase superfamily

Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase

Diamine oxidase (DAO) is an enzyme involved in the regulation of cell proliferation and the immune response. This enzyme performs oxidative deamination in the catabolism of biogenic amines, including, among others, histamine, putrescine, spermidine, and spermine. The mechanistic details underlying the reductive half-reaction of the DAO-catalyzed oxidative deamination which leads to the reduced enzyme cofactor and the aldehyde product are, however, still under debate. The catalytic mechanism was proposed to involve a prototropic shift from the substrate–Schiff base to the product–Schiff base, which includes the rate-limiting cleavage of the Cα–H bond by the conserved catalytic aspartate. Our detailed mechanistic study, performed using a combined quantum chemical cluster approach with empirical valence bond simulations, suggests that the rate-limiting cleavage of the Cα–H bond involves direct hydride transfer to the topaquinone cofactora mechanism that does not involve the formation of a Schiff base. Additional investigation of the D373E and D373N variants supported the hypothesis that the conserved catalytic aspartate is indeed essential for the reaction; however, it does not appear to serve as the catalytic base, as previously suggested. Rather, the electrostatic contributions of the most significant residues (including D373), together with the proximity of the Cu²⁺ cation to the reaction site, lower the activation barrier to drive the chemical reaction

The Alkaline Hydrolysis of Sulfonate Esters: Challenges in Interpreting Experimental and Theoretical Data

Sulfonate ester hydrolysis has been the subject of recent debate, with experimental evidence interpreted in terms of both stepwise and concerted mechanisms. In particular, a recent study of the alkaline hydrolysis of a series of benzene arylsulfonates (Babtie et al., <i>Org. Biomol. Chem.</i> <i>10</i>, <b>2012</b>, 8095) presented a nonlinear Brønsted plot, which was explained in terms of a change from a stepwise mechanism involving a pentavalent intermediate for poorer leaving groups to a fully concerted mechanism for good leaving groups and supported by a theoretical study. In the present work, we have performed a detailed computational study of the hydrolysis of these compounds and find no computational evidence for a thermodynamically stable intermediate for any of these compounds. Additionally, we have extended the experimental data to include pyridine-3-yl benzene sulfonate and its <i>N</i>-oxide and <i>N</i>-methylpyridinium derivatives. Inclusion of these compounds converts the Brønsted plot to a moderately scattered but linear correlation and gives a very good Hammett correlation. These data suggest a concerted pathway for this reaction that proceeds via an early transition state with little bond cleavage to the leaving group, highlighting the care that needs to be taken with the interpretation of experimental and especially theoretical data

Data of theoretical modelling of epigenetically modified DNA sequences

<p>Data file 1<br>Local base pair parameters for the central GC in d(GCG) and d(ACA) modifications. The shear, stretch and stagger parameters are measured in Å, and the buckle, propeller and opening parameters are measured in ˚.</p> <p>Data file 2<br>Base pair step and local helical parameters for the central GC in d(GCG) and d(ACA) modifications. The slide and X-displacement parameters are measured in Å, and the roll, twist and inclination parameters are measured in ˚.</p> <p>Data file 3<br>Comparison between the obtained base pair parameters for the d(GCG) using different levels of theory (DFT and the QM/MM with 2bps or 6bps in the high level layer). The shear, stretch and stagger parameters are measured in Å, and the buckle, propeller and opening parameters are measured in ˚.</p> <p>Data file 4<br>Comparison between the obtained base step parameters for the d(GCG) using different levels of theory (DFT and the QM/MM with 2bps or 6bps in the high level layer). The slide, shift and rise parameters are measured in Å, and the tilt, roll and twist parameters are measured in ˚.</p> <p>Data file 5<br>Values of local base pair parameters of central GC pair of d(GCG) obtained after energy minimization. The shear, stretch and stagger parameters are measured in Å, and the buckle, propeller and opening parameters are measured in ˚.</p> <p>Data file 6<br>Values of base pair step parameters of d(GCG), obtained after energy minimization. The shift, slide and rise parameters are measured in Å, and the tilt, roll and twist parameters are measured in °.</p> <p>Data file 7<br>Cartesian coordinates of key species.</p> <p> </p

Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation

<div><p>We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.</p></div

Base-Catalyzed Dehydration of 3‑Substituted Benzene <i>cis</i>-1,2-Dihydrodiols: Stabilization of a Cyclohexadienide Anion Intermediate by Negative Aromatic Hyperconjugation

Evidence that a 1,2-dihydroxycyclohexadienide anion is stabilized by aromatic “negative hyperconjugation” is described. It complements an earlier inference of “positive” hyperconjugative aromaticity for the cyclohexadienyl cation. The anion is a reactive intermediate in the dehydration of benzene <i>cis</i>-1,2-dihydrodiol to phenol. Rate constants for 3-substituted benzene <i>cis</i>-dihydrodiols are correlated by σ^– values with ρ = 3.2. Solvent isotope effects for the reactions are <i>k</i>_H₂O/<i>k</i>_D₂O = 1.2–1.8. These measurements are consistent with reaction via a carbanion intermediate or a concerted reaction with a “carbanion-like” transition state. These and other experimental results confirm that the reaction proceeds by a stepwise mechanism, with a change in rate-determining step from proton transfer to the loss of hydroxide ion from the intermediate. Hydrogen isotope exchange accompanying dehydration of the parent benzene <i>cis</i>-1,2-dihydrodiol was not found, and thus, the proton transfer step is subject to internal return. A rate constant of ∼10¹¹ s^–1, corresponding to rotational relaxation of the aqueous solvent, is assigned to loss of hydroxide ion from the intermediate. The rate constant for internal return therefore falls in the range 10¹¹–10¹² s^–1. From these limiting values and the measured rate constant for hydroxide-catalyzed dehydration, a p<i>K</i>_a of 30.8 ± 0.5 was determined for formation of the anion. Although loss of hydroxide ion is hugely exothermic, a concerted reaction is not enforced by the instability of the intermediate. Stabilization by negative hyperconjugation is proposed for 1,2-dihydroxycyclohexadienide and similar anions, and this proposal is supported by additional experimental evidence and by computational results, including evidence for a diatropic (“aromatic”) ring current in 3,3-difluorocyclohexadienyl anion