Methotrexate and folate binding to dihydrofolate reductase. Separate characterization of the pteridine and p-aminobenzoyl binding sites by resonance Raman spectroscopy.
By using 324- and 350.6-nm excitation, it is possible to obtain selectively the resonance Raman spectra of the p-aminobenzoyl and pteridine chromophores, respectively, within methotrexate (MTX) or folate. Thus, for a single ligand, by changing the wavelength for excitation, the geometric conformations of both chromophores can be monitored separately. Resonance Raman spectra are reported for MTX bound to dihydrofolate reductases from Escherichia coli and from Lactobacillus casei, in each case in the presence and absence of NADPH. Additionally, some data are presented for enzyme-bound folate. The resonance Raman data support the conclusions of other workers that MTX binds with its pteridine ring protonated while the pteridine ring within folate is bound as a neutral species. However, for MTX, marked differences exist between the electronic distribution in the protonated pteridine ring for the ligand free in solution and for the bound species. The rearrangement of the pteridine electrons over and above that accompanying protonation explains the absorption properties of bound MTX, and together with protonation may account in part for the high affinity of MTX for the enzyme. The resonance Raman spectra show that slight differences exists between the pteridine sites for MTX in the three dihydrofolate reductases studied while no differences could be detected among the p-aminobenzoyl sites. In each MTX-protein complex, however, there appears to be a marked change in the geometry of the amide group in the benzoyl linkage of MTX compared to the geometry found in the free ligand. The resonance Raman spectra of MTX bound to the enzymes were unchanged upon the addition of the cofactor NADPH, indicating that the cofactor does not bring about marked electron rearrangement in the bound ligands.
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005492:Folic Acid; D008727:Methotrexate; D011485:Protein Binding; D011621:Pteridines; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D011621:Pteridines; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate