The carcinogenesis of urethane (ethyl carbamate), a byproduct of fermentation that is consistently found in various food products, was investigated with a combination of kinetic experiments and quantum chemical calculations. The main objective of the study was to find ΔGa, the activation free energy for the rate-limiting step of the SN2 reaction among the ultimate carcinogen of urethane, vinyl carbamate epoxide (VCE), and different nucleobases of the DNA. In the experimental part, the second-order reaction rate constants for the formation of the main 7-(2-oxoethyl)guanine adduct in aqueous solutions of deoxyguanosine and in DNA were determined. A series of ab initio, density functional theory (DFT), and semiempirical molecular orbital (MO) calculations was then performed to determine the activation barriers for the reaction between VCE and nucleobases methylguanine, methyladenine, and methylcytosine. Effects of hydration were incorporated with the use of the solvent reaction field method of Tomasi and coworkers and the Langevine dipoles model of Florian and Warshel. The computational results for the main adduct were found to be in good agreement with the experiment, thus presenting strong evidence for the validity of the proposed SN2 mechanism. This allowed us to predict the activation barriers of reactions leading to side products for which kinetic experiments have not yet been performed. Our calculations have shown that the main 7-(2-oxoethyl)deoxyguanosine adduct indeed forms preferentially because the emergence of other adducts either proceeds across a significantly higher activation barrier or the geometry of the reaction requires the Watson–Crick pairs of the DNA to be broken. The computational study also considered the questions of stereoselectivity, the ease of the elimination of the leaving group, and the relative contributions of the two possible reaction paths for the formation of the 1,N2-ethenodeoxyguanosine adduct.
COBISS.SI-ID: 5643802
Molecular dynamics simulations with separate thermostats for rotational and translational motion were used to study the effect of these degrees of freedom on the structure of water around model solutes. To describe water molecules we used the SPC/E model. The simplest solute studied here, the hydrophobe, was represented as a Lennard-Jones particle. Since direct interaction between the hydrophobe and water molecules has no angular dependence the influence of the increase of the rotational temperature on the solvation of a hydrophobe is only indirect. In the next step the central solute was assumed to be charged with either a positive or a negative charge to mimic an ion in water. Hence, depending on the charge of the ion, the neighboring water molecules assumed different angular distributions. The principal conclusions of this work are: (i) an increase of the translational temperature always decreases the height of the first peak in the solute-water radial distribution function; (ii) an increase of the rotational temperature yields an increase in the first peak in the solute-water radial distribution function for hydrophobes and cations; (iii) in contrast to this, the solvation peak decreases around ions with sufficiently large negative charge; and (iv) an increase of the rotational temperature affects cations in an opposite way to anions. For this reason complex molecules with a small net charge may not be very sensitive to variation of the rotational temperature.
COBISS.SI-ID: 1708079
Generally, enzymes are perceived as being specific for both their substrates and the reaction they catalyze. This standard paradigm started to shift and currently enzyme promiscuity towards various substrates is perceived rather as the rule than the exception. Enzyme promiscuity seems to be vital for proteins to acquire new functions, and therefore for evolution itself. The driving forces for promiscuity are manifold and consequently challenging to study. Binding free energies, which can be calculated from computer simulations, represent a convenient measure for them. Here, we investigate the binding free energies between the enzyme pyranose dehydrogenase (PDH) and a sugar-substrate by computational means. PDH has an extraordinarily promiscuous substrate-specificity, making it interesting for e.g. bioelectrochemical applications. By introducing modifications to the sugar-structure used for the molecular dynamics simulations, we could simultaneously study all 32 possible aldohexopyranoses from a single simulation. This saves costly computational resources and time for setting up and analyzing the simulations. We could nicely reproduce experimental results and predict so far undetected sugar-oxidation products, directing further experiments. This study gives novel insights into PDH's substrate promiscuity and the enzyme's applicability. A similar approach could be applied to study the promiscuity of other enzymes.
COBISS.SI-ID: 5613338