Conformational changes induced in dihydrofolate reductase by folates, pyridine nucleotide coenzymes, and methotrexate.
Dihydrofolate reductase (EC 1.5.1.3; 5,6,7,8-tetrahydrofolate:NADP(+) oxidoreductase) from antifolate-resistant Lactobacillus casei has been isolated in pure form and examined in solution by high resolution proton magnetic resonance spectroscopy. The 220 MHz proton magnetic resonance spectrum of this small enzyme (about 15,000 daltons) consists of several distinct resonance peaks that provide a sensitive nonperturbing probe of its conformational state. Comparison of catalytically active enzyme with preparations denatured in 6 M urea demonstrates dramatically the overall contribution of secondary and tertiary structure to its proton magnetic resonance spectra. More subtle differences existing among several catalytically active enzyme forms may also be readily differentiated by proton magnetic resonance spectroscopy, e.g., the spectra of the ligand-free enzyme and forms containing stoichiometric amounts of tightly bound folate and dihydrofolate, each obtained separately by affinity chromatography, are easily identified. Addition of ligands to these spectroscopically distinct forms may induce changes in their proton magnetic resonance spectra. For example, addition of equimolar dihydrofolate to the apoenzyme converts its relatively featureless aromatic proton magnetic resonance spectrum to one indistinguishable from that of the original enzyme-dihydrofolate binary complex obtained chromatographically. Interaction of the pyridine nucleotide coenzymes NADP(+) or NADPH or of the antifolate Methotrexate with apoenzyme induces additional distinct spectral changes. Enzyme-NADPH and enzyme-Methotrexate binary complexes, which have different aromatic region proton magnetic resonance spectra, are converted to ternary complexes having quite similar spectra by addition of Methotrexate and NADPH, respectively, thus suggesting that an ordered addition of ligands is not required.
D005492:Folic Acid / Q000494:pharmacology*; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification; D014508:Urea
D008024:Ligands; D009249:NADP; D014508:Urea; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate