Functional Role of Aspartic Acid-27 in Dihydrofolate Reductase Revealed by Mutagenesis

Abstract

The crystal structures and enzymic properties of two mutant dihydrofolate reductases ( Escherichia coli ) were studied in order to clarify the functional role of an invariant carboxylic acid (aspartic acid at position 27) at the substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp 27 with asparagine; the other is a primary-site revertant to Ser 27 . The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing p H. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp 27 participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.