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

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dihydrofolate reductase, in which all the aromatic protons except the 2,6-protons of the tyrosine residues have been replaced by deuterium, has been prepared from Lactobacillus casei grown on a mixture of normal and deuterated amino acids.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 degrees rotation about the C$_{\beta}$-C$_{\gamma}$ 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-diaminopyrimidine complex is found to be tighter than that to the enzyme alone.