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.