The major objective of this study is to investigate the mode of binding of substrates and various chemotherapeutic agents to the enzymes thymidylate synthase and dihydrofolate reductase, using primarily proton nuclear magnetic resonance. The enzymes used in this study will be isolated from Lactobacillus casei cells grown in completely deuterated or selectively deuterated media. The completely deuterated and selectively deuterated enzymes have relatively simple proton NMR spectra, therefore it will be possible to observe and assign proton resonances of enzyme-bound drugs and other ligands. Hydrogen will be introduced at specific positions in the enzymes and other proteins by growing L. casei in deuterated media that contain specific protio amino acids. The proton labels in these proteins will serve as NMR """"""""probes"""""""" that are sensitive to conformational changes induced by the binding of ligands to the enzymes. When possible, the proton resonances of IH-labeled enzymes will be assigned to specific positions in the amino acid sequence. Changes in the chemical shifts of the assigned resonances brought about by the binding of ligands should provide valuable insight into the mechanisms by which these enzymes bind substrates and inhibitors. The fully deuterated enzymes will be used to assign and determine the chemical shifts of resonances from enzyme-bound IH-ligands, which should also provide information concerning the mechanisms of binding. Some of the techniques that will be developed and tested in this study may be of general utility in the study of biomolecular mechanisms. Deuterated L. casei cells could provide other important deuterated biological molecules that are amenable to studies using powerful techniques such as NMR, neutron scattering, and IR. Recombinant DNA techniques may even provide deuterated mammalian proteins that could be used in studies similar to these.
