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Determination of Allosteric Residues Involved in Ligand Selectivity in the Dihydrofolate Reductase Family

Dihydrofolate Reductase (DHFR) is an essential enzyme for most organisms, ranging from bacteria to humans. It has essential functions in DNA biosynthesis and cell replication and cell growth can be diminished by inhibition of DHFR. It is critical to understand inhibitor specificity of DHFR homologs from different organisms. To design compounds that inhibit DHFR from infectious organisms while leaving human DHFR alone. A computational analysis was previously developed to predict residues away from the active site that are significant in ligand specificity in the DHFR enzyme family. Allosteric residues H38, A39, I86, W85, Q84, T125, F126, and Y127 (human DHFR numbering) were predicted to be key residues in ligand specificity in the DHFR enzyme family. To validate these predictions, mutations (H38N, A39Y, I86A, T125A, F126L, and Y127A) have been introduced into the B. Strearothermophilus DHFR gene and the mutants have been expressed and purified. We have measured the binding constants to select ligands (Trimethoprim, Raltitrexed, and Pyrimethamine) by equilibrium binding titrations using Fluoromax‐4. Comparisons on the ligand binding profiles of the mutants to those of the wild type enzyme have revealed that some of the mutated amino acid positions do have an effect on ligand selectivity and this work is still going on. Turnover (kcat) values have also been determined for the mutants and wildtype DHFR and showed that mutations do not affect catalytic activity, with the exception of the I86A mutant. Further comparison of the ligand specificity of double mutants with two mutated positions is currently in progress to investigate synergy between different positions in ligand binding specificity in this enzyme family.