1 H nuclear magnetic resonance studies of the tyrosine residues of selectively deuterated Lactobacillus casei dihydrofolate reductase

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Abstract
A selectively deuterated dihydrofolate reductase, in which all the aro­matic protons except the 2, 6-protons of the tyrosine residues have been replaced by deuterium, has been prepared from Lactobacillus casei grown on a mixture of normal and deuterated amino acids. The aromatic region of the 1 H n. m. r. spectrum of this enzyme contains only resonances from the five tyrosine residues. For each tyrosine, the 2- and 6-protons have the same chemical shift, indicating rapid interconversion of the two conformers related by 180° rotation about the C β -C γ bond. The effects of sub­strate, inhibitor and coenzyme binding on the tyrosine residues have been investigated; four of the five residues are affected by ligand binding. Using the weakly binding ligands 2, 4-diaminopyrimidine and p -nitrobenzoyl-l-glutamate to connect the spectra of the free enzyme with those of the complexes, it is possible to give a detailed description of the effects of ligand binding on individual residues. In the binary complexes, methotrexate affects three tyrosine residues, only one of which is affected by folate, indicating a significant difference in the mode of binding of substrates and inhibitors. The co-enzymes NADP + and NADPH lead to broadly similar changes in the spectrum, except for one resonance which is shifted in opposite directions by the two co-enzymes. The oxidized and reduced co­enzymes also differ in their effects on the changes produced by inhibitor binding; the spectrum of the enzyme-NADPH-methotrexate complex is similar to that of the enzyme-methotrexate complex, while that of the enzyme-NADP + -methotrexate complex is not. In contrast to the be­haviour seen in the binary complexes, the spectrum of the enzyme-NADP + -folate complex is very similar to that of enzyme-NADP + -methotrexate. Evidence is presented that some, at least, of the changes in chemical shift of the tyrosine residues are due to ligand-induced conformational changes. The binding of p -nitrobenzoyl-l-glutamate to the enzyme-2, 4-diamino-pyrimidine complex is found to be tighter than that to the enzyme alone.

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