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

Abstract

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dihydrofolate reductase, in which all the aro­matic protons except the 2, 6-protons of the tyrosine residues have been replaced by deuterium, has been prepared from Lactobacillus casei grown on a mixture of normal and deuterated amino acids.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 1H 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 Cβ-Cγ 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.