Abstract

The dihydrofolate reductase of Escherichia coli is rapidly and irreversibly inactivated in the presence of the proteolytic enzyme Pronase. However, the reductase is almost completely protected when dihydrofolate, NADP, NADPH, or N⁵-formyltetrahydrofolate are present at saturating concentrations. NAD, NADH, 2-amino-4-hydroxy-6hydroxymethyl dihydropteridine, p-aminobenzoylglutamate, amid glutamate were all ineffective in this regard. On the other hand, NAD did not accelerate the inactivation of E. coli dihydrofolate reductase by Pronase. Certain small-molecule, heterocyclic inhibitors of dihydrofolate reductase were also excellent protective agents when added at 30 times the concentration needed for 50% inhibition of the reductase. Surprisingly, at this same concentration certain other inhibitors with equal or better capacity for binding to the reductase failed to show any protective activity. The structural requirements for protection were explored, and some possible implications of the findings are discussed.