The intensity changes of proton NMR resonances of NADPH and NADP+ were employed to monitor the oxidation of NADPH in the presence of dihydrofolate reductase (DHFR) from Escherichia coli. It was found that in addition to the normal reductase activity E. coli dihydrofolate reductase (DHFR) also catalyzes oxygen-dependent oxidation of NADPH to NADP+ in the absence of folate or dihydrofolate. Mutant-type DHFR (Asp27→Asn) shows almost no activity at pH 7.0, while at pH 4.0 it regains its activity. This enzymatic activity is characterized by a fast initial rate followed by a gradual leveling-off (slow phase), which appears to be due to the depletion of oxygen in the solution, under our experimental conditions. From the NADP+ concentration at the fast phase we estimated that four moles of NADPH were oxidized per mole of oxygen consumed. The specific activity, determined from the initial rate of NADPH oxidation, is highly pH dependent. It decreases from 9.6 × 10-3 units/mg protein at pD = 5.2 to 0.56 × 10-3 units/mg at pD = 8.0 at 21 °C. The pH dependence fits quite well to a titration curve of a single group with pKa = 6.5. DHFR inhibitors, trimethoprim (TMP) and methotrexate (MTX) completely inhibit this activity, whereas folate and H4F do not. NADP+ appears to partially inhibit this reaction. Thus this oxidative activity of DHFR shares several similar characteristics of the normal reductase activity and suggests some common structural requirements.
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