Dihydrofolate reductase: multiple conformations and alternative modes of substrate binding.
The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent [Birdsall, B., Gronenborn, A. M., Hyde, E. I., Clore, G. M., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1982) Biochemistry 21, 5831]. The assignment of the resonances of all the aromatic protons of the ligand molecules in all three conformational states of the complex has now been completed by using a variety of NMR methods, particularly two-dimensional exchange experiments. The resonances of the nicotinamide protons of the coenzyme and the pteridine 7-proton of the folate have different chemical shifts in the three conformations, in some cases differing by more than 1 ppm. Comparison of the COSY spectra of the complex at low pH (conformation I) and high pH (conformations IIa and IIb) with that of the enzyme-methotrexate-NADP+ complex shows only slight differences in the conformation of the protein. The pattern of chemical shift changes in the ligand and the protein indicates that the structural differences are localized within the active site of the enzyme. Nuclear Overhauser effects (NOEs) are observed between the nicotinamide 5- and 6-protons and the methyl resonance of Thr 45 at both low and high pH, indicating that there is no major movement of the nicotinamide ring. By contrast, NOEs are observed between the pteridine 7-proton and the methyl protons of Leu 19 and Leu 27 in conformations I and IIa but not in conformation IIb.(ABSTRACT TRUNCATED AT 250 WORDS)
Birdsall
B
B
Division of Physical Biochemistry, National Institute for Medical Research, London, U.K.
Feeney
J
J
Tendler
S J
SJ
Hammond
S J
SJ
Roberts
G C
GC
D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000378:metabolism; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases; D009249:NADP / Q000378:metabolism; D009536:Niacinamide; D011487:Protein Conformation; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D008024:Ligands; D009536:Niacinamide; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D008727:Methotrexate