Inhibitor binding analysis of dihydrofolate reductases from various species.

Dihydrofolate reductases have been purified approximately 100-fold from Escherichia coil, Staphylococcus aureus, and Proteus vulgaris and to a lesser degree from rat, rabbitr guinea pig, and human liver. Average Michaelis constants of 2.3 x 10-5 M for dihydrofolic acid and 1.8 x 10-5 M for NADPH have been obtained for the three bacterial enzymes. NADPH is the preferred reductant but could be replaced by NADH with about 25% efficiency. pH-activity curves, in the case of the bacterial enzymes, show a single peak with maximum activity between pH 6.8 and 7.2. Strong correlations have been found between the binding of a drug by a particular dihydrofolate reductase and the capacity of that drug to inhibit the source organism in vitro. In addition, each dihydrofolate reductase was shown to possess a pattern of inhibition that is distinct for the species under investigation. These differences between and among bacterial and mammalian reductases may be due to changes in amino acid composition at the active sites.