Dihydrofolate reductase from trimethoprim-resistant Escherichia coli MB 3746 and MB 3747. Purification, amino acid composition, and some kinetic properties.

Dihydrofolate reductase has been isolated and purified to homogeneity in g6od yield from two trimethoprim-resistant strains of Escherichia coli K12, strains MB 3746 and MB 3747. The two enzymes are closely related to one another and to the dihydrofolate reductase from E. coli MB 1428. The 3746 and 3747 reductases both exhibit an apparent molecular weight of 17,500 with almost identical amino acid compositions. The respective turnover numbers in moles of dihydrofolate reduced per min per mol of enzyme at pH 7.2 and 25°C are 630 and 740 for the 3746 and 3747 enzymes; the respective Michaelis constants for dihydrofolate are 1.6 and 7.3 PM, and the respective inhibition constants for trimethoprim are 1.1 and 11 nM. The pH at which maximal enzyme activity is observed is 8.0 and below 6.4 for the respective enzymes. Thus, the 3747 reductase is like mammalian reductase in two respects: a lowered affinity for trimethoprim and maximal activity well below neutral pH. The pmr spectra at 300 MHz of the binary methotrexate complexes of the 1428, 3746, and 3747 reductases are similar. The pK’ values at 25°C of the 5 histidine residues are 8.2, 7.5, 6.9, 6.4, and 5.9 for the binary 3746 reductase l methotrexate complex and 8.2, 7.3, 6.7, 6.0, and 7.6 for the binary 3747 reductasee methotrexate complex. The reactivity of the 3746 reductase and its complexes with folate, dihydrofolate, methotrexate, and NADPH, with the histidine-specific reagent ethoxyformic anhydride, show that the 3746 and 1428 enzymes have similar, but not identical, histidine environments.