Reaction mechanism of caspases: insights from QM/MM Car-Parrinello simulations.

Caspases are fundamental targets for pharmaceutical interventions in a variety of diseases involving disregulated apoptosis. Here, we present a quantum mechanics/molecular mechanics Car-Parrinello study of key steps of the enzymatic reaction for a representative member of this family, caspase-3. The hydrolysis of the acyl-enzyme complex is described at the density functional (BLYP) level of theory while the protein frame and solvent are treated using the GROMOS96 force field. These calculations show that the attack of the hydrolytic water molecule implies an activation free energy of ca. DeltaF(A) approximately equal 19 +/- 4 kcal/mol in good agreement with experimental data and leads to a previously unrecognized gem-diol intermediate that can readily (DeltaF(A) approximately equal 5 +/- 3 kcal/mol) evolve to the enzyme products. Our findings assist in elucidating the striking difference in catalytic activity between caspases and other structurally well-characterized cysteine proteases (papains and cathepsins) and may help design novel transition-state analog inhibitors.

[A new proposal for nutritional education in a group of obese adolescents]. / Una nuova proposta di educazione alimentare in un gruppo di adolescenti obesi.

A new "active" system of monitoring and dietary education was experimented in a group of 20 obese adolescents waiting to start diet therapy. Each subject was requested to record all food and drink consumed during two separate periods, each lasting 7 days, at a distance of 15 days from each other, at home using an optic reader and book of bar codes. On the basis of answers to a questionnaire which was completed at the beginning and end of the study, aimed at assessing the level of knowledge of basic food hygiene, the majority of participants considered the experience useful and amusing and were willing to repeat it; in addition, a greater knowledge of nutritional principles and of the rations consumed was shown at the end of the study. The results of the study were analysed using the Food Meter-Miles computerised system and showed daily calorie intakes of 1514 +/- 524.0, M +/- DS (protein 18.3 +/- 5.1%, lipids 33.7 +/- 6.2%, glucose 47.9 +/- 8.6%, total fibre 14.4 +/- 4.6 g) in female subjects, and 2096.1 +/- 80.8 (protein 16.1 +/- 3.6%, lipids 38.2 +/- 2.9%, glucose 45.7 +/- 3.9%, total fibre 18.1 +/- 2.0 g) in male subjects. The number of foods chosen was very low considering the range of foods available (277) and the length of time studied: 35.7 +/- 11.8 and 35.0 +/- 6.0 respectively for female and male subjects. With regard to the number and type of meals eaten, a high number of snacks was observed which supplemented or replaced main meals.

Molecular dynamics studies of caspase-3.

Caspase-3 is a fundamental target for pharmaceutical interventions against a variety of diseases involving disregulated apoptosis. The enzyme is active as a dimer with two symmetry-related active sites, each featuring a Cys-His catalytic dyad and a selectivity loop, which recognizes the characteristic DEVD pattern of the substrate. Here, a molecular dynamics study of the enzyme in complex with two pentapeptide substrates DEVDG is presented, which provides a characterization of the dynamic properties of the active form in aqueous solution. The mobility of the substrate and that of the catalytic residues are rather low indicating a distinct preorganization effect of the Michaelis complex. An essential mode analysis permits us to identify coupled motions between the two monomers. In particular, it is found that the motions of the two active site loops are correlated and tend to steer the substrate toward the reactive center, suggesting that dimerization has a distinct effect on the dynamic properties of the active site regions. The selectivity loop of one monomer turns out to be correlated with the N-terminal region of the p12 subunit of the other monomer, an interaction that is also found to play a fundamental role in the electrostatic stabilization of the quaternary structure. To further characterize the specific influence of dimerization on the enzyme essential motions, a molecular dynamics analysis is also performed on the isolated monomer.

The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study.

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.