Interconvertible forms of Escherichia coli dihydrofolate reductase with different affinities for analogs of dihydrofolate.
Dihydrofolate reductase from Escherichia coli exists as two species, which show large differences in their affinities for trimethoprim and for pyrimethamine. The two species are present in approximately equal proportions. Each possesses one binding site per mol with dissociation constants (KD) of 14 and 1400 nM, respectively, for the binding of trimethoprim in the binary complex, and of 5 and 47 nM for the pyrimethamine binary complex. In the formation of the ternary complex with NADPH, trimethoprim bound to dihydrofolate reductase as if all the enzyme existed as a single species with a KD for trimethoprim of 1.9 nM. Formation of the trimethoprim NADPH ternary complex thus involves strong cooperative effects, and interconversion of the two species. The binding of pyrimethamine in the ternary complex was indistinguishable from its binding in the binary complex, showing neither the cooperative effects, nor the interconversion of the two species observed with trimethoprim. The species with a low KD for trimethoprim and pyrimethamine could be isolated by selective proteolysis. It was quite stable, but could be converted to a mixture of the original species via formation of the ternary complex with trimethoprim, as predicted from the binding data. The results are interpreted in terms of a model in which the two species can be interconverted only via the formation of a common ternary complex, which can be formed after the binding of trimethoprim or dihydrofolate, but not pyrimethamine. The model is shown to be consistent with all data from the measurements of both binding and inhibition by both ligands.
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D011402:Pronase; D011485:Protein Binding; D011739:Pyrimethamine / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D007527:Isoenzymes; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D011402:Pronase; D011739:Pyrimethamine