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 Asp27 with asparagine; the other is a primary-site revertant to Ser27. The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing pH. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp27 participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.

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