Circular dichroism studies of dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428: ternary complexes.
Circular dichroism has been used to monitor the binding of pyridine nucleotide cofactors to enzyme-folate analog complexes of dihydrofolate reductase from Escherichia coli B (MB 1428). The enzyme binds one molar equivalent of many folate analogs and two molar equivalents of several pyridine nucleotide cofactors. The apo-enzyme has very low optical activity. The binding of folate analogs including folate, dihydrofolate, methotrexate, trimethoprim and pyrimethamine induce large Cotton effects. Pyridine nucleotides when bound to the enzyme-folate analog complexes also induce new optically active bands; all the effects being due to the first molar equivalent of cofactor bound. NADPH and NADP+ induce very similar bands when bound to the enzyme-methotrexate complex suggesting that the geometry of the complexes formed are very similar. The oxidized and reduced cofactor likewise have similar effects on the enzyme-folate complex. However, NADPH and NADP+ addition to both the enzyme-trimethoprim and enzyme-pyrimethamine complexes have significantly different effects on the circular dichroism spectra, suggesting that the inhibitors which are less homologous to the natural dihydrofolate substrate allow more conformational freedom in the enzyme-inhibitor-cofactor complex. In most cases the prior binding of the folate analog greatly increases the binding of the first molar equivalent of cofactor so that at concentrations of approx. 5-20 muM the binding appears stoichiometric. Pyrimethamine is an exception in that it apparently has no effect on the binding of NADPH to the enzyme.
D000247:Adenosine Diphosphate Sugars; D001665:Binding Sites; D002942:Circular Dichroism; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005492:Folic Acid / Q000031:analogs & derivatives; D008727:Methotrexate / Q000494:pharmacology; D009243:NAD; D009249:NADP / Q000031:analogs & derivatives; D009537:Nicotinamide Mononucleotide; D011485:Protein Binding; D011487:Protein Conformation; D012266:Ribose; D013056:Spectrophotometry, Ultraviolet; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D000247:Adenosine Diphosphate Sugars; D009243:NAD; D009537:Nicotinamide Mononucleotide; D009249:NADP; D012266:Ribose; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate