α-Pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase

Abstract

The α epimers of pyridine nucleotides are almost totally inactive as reductants in dehydrogenase reactions. In contrast, the R plasmid R67-specified dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) isolated from trimethoprim-resistant Escherichia coli utilized α-NADPH and α-NADH in addition to the 'normal' β-epimers. The enzymes from bacterial and mammalian sources used only β-NADPH and β-NADH. The K(m) value for α-NADPH (16 μM) was 4-fold greater than that for β-NADPH (4 μM), while the maximal velocity of the α-NADPH-catalyzed reaction was 70% of that seen with the β-NADPH. β-NADP+ and α-NADP+ were competitive inhibitors of the R67 enzyme. Pyridine nucleotide analogues such as deamino- and acetyl-NADPH were used readily by bacterial, plasmid, and mammalian enzymes, whereas thio-NADPH was used only by the plasmid enzyme. These data suggest that the enzyme from R plasmid R67 possesses a pyridine nucleotide binding site different from that of other dihydrofolate reductases and dehydrogenases.