Computer Simulation of Protein Tyrosine Phosphatase Reaction Mechanisms and Dihydrofolate Reductase Inhibition

Protein tyrosine phosphatases catalyse the hydrolysis of phosphotyrosine residues in proteins, which is an important reaction in the cell signalling system. The three dimensional structure of such a protein tyrosine phosphatase has been used in a computational study of the reaction mechanism at the atomic level. Free energy calculations of different reaction pathways were performed using the empirical valence bond method in combination with free energy perturbation and molecular dynamics simulations. The objective was to find a reaction mechanism that is compatible with experimental data and to elucidate the specific interactions important for catalysis. The free energy calculations of the simulated reaction in the solvated enzyme–substrate complex correctly reproduce the observed reaction rates for wild type and mutant enzymes. However, the results show that a different reaction mechanism is energetically more plausible than that previously proposed. The difference pertains to the ionisation state of the enzyme–substrate complex. This mechanism is found to be compatible with enzymological and structural data from earlier studies of protein tyrosine phosphatases. Molecular dynamics simulations of a cdc25 phosphatase reveal that this enzyme has to undergo a conformational change in association with substrate binding in order to efficiently catalyse the phosphate hydrolysis reaction. The predicted change in the protein structure has later been confirmed by X-ray crystallography. Kinetic isotope effects are often used to investigate possible reaction mechanisms in phosphoryl transfer processes in enzymes and solution. Quantum mechanical calculations of heavy atom isotope effects on phosphate and phosphate esters demonstrate the importance of a realistic representation of the surrounding solvent. Calculations with a dielectric continuum are not adequate because the vibrational coupling to the solvent molecules has to be included in order to accurately reproduce the experimentally measured isotope effects. Computational studies of enzyme–inhibitor complexes were also conducted as a part of a multi-disciplinary drug design project. The linear interaction energy method as well as empirical scoring functions were used to predict the affinity and species selectivity of novel lipophilic inhibitors of the enzyme dihydrofolate reductase. The aim was to design compounds that preferentially inhibit dihydrofolate reductase from Pneumocystis carinii and not the corresponding human enzyme, which turns out to be a challenging tas