title: Novel mechanism-based substrates of dihydrofolate reductase and the thermodynamics of ligand binding: a comparison of theory and experiment for 8-methylpterin and 6,8-dimethylpterin.
abstract: Molecular dynamics simulation and free energy perturbation techniques have been used to study the relative binding free energies of the designed mechanism-based pterins, 8-methylpterin and 6,8-dimethylpterin, to dihydrofolate reductase (DHFR), with cofactor nicotinamide adenine dinucleotide phosphate (NADPH). The calculated free energy differences suggest that DHFR.NADPH.6,8-dimethylpterin is thermodynamically more stable than DHFR.NADPH.8-methylpterin by 2.4 kcal/mol when the substrates are protonated and by 1.3 kcal/mol when neutral. The greater binding strength of 6,8-dimethylpterin may be attributed largely to hydration effects. In terms of an appropriate model for the pH-dependent kinetic mechanism, these differences can be interpreted consistently with experimental data obtained from previous kinetic studies, i.e., 6,8-dimethylpterin is a more efficient substrate of vertebrate DHFRs than 8-methylpterin. The kinetic data suggest a value of 6.6 +/- 0.2 for the pKa of the active site Glu-30 in DHFR.NADPH. We have also used experimental data to estimate absolute values for thermodynamic dissociation constants of the active (i.e., protonated) forms of the substrates: these are of the same order as for the binding of folate (0.1-10 microM). The relative binding free energy calculated from the empirically derived dissociation constants for the protonated forms of 8-methylpterin and 6,8-dimethylpterin is 1.4 kcal/mol, a value which compares reasonably well with the theoretical value of 2.4 kcal/mol.
authors.affiliation: Department of Biochemistry, University of Sydney, N.S.W., Australia.
authors.forename: P L
authors.initials: PL
authors.lastname: Cummins
authors.forename: J E
authors.initials: JE
authors.lastname: Gready
mesh_terms: D003198:Computer Simulation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D015394:Molecular Structure; D011485:Protein Binding; D011622:Pterins / Q000737:chemistry / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008024:Ligands; D011622:Pterins; C080386:8-methylpterin; C080384:6,8-dimethylpterin; D013762:Tetrahydrofolate Dehydrogenase