The goal of this research is to elucidate the chemical mechanism of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (CMPDH). Because CMPDH is an enzyme peculiar to the de novo biosynthesis of tyrosine, a pathway which is not present in humans, an understanding of its mechanism can lead to the development of new antibiotics, bacteriostatics, or herbicides. The long term objective is to use the knowledge of the CMPDH reaction mechanism to aid in design of new antibiotics which can be used topically in the oral cavity to limit the growth of the oral pathogens responsible for plaque, caries, and periodontal disease which is a growing problem in an aging population. 13C NMR will be applied for the first time to investigate the mechanism of CMPDH. 13C NMR will allow observation of ligands (substrates, products, reaction intermediates) bound at the active site of the enzyme. 13C labelled substrates will be prepared, predominantly by established enzymological methods, and their NMR spectra both free in solution and when bound to the enzyme will be obtained. The effect of the enzyme on the chemical shifts and coupling constants of the 13C labelled ligands will reveal the structures of the substrates, products, and reaction intermediates bound at the active site. A thorough characterization of the bound ligands will be obtained through use of five different 13C labelled substrates, each labelled in two to four different positions with 13C. Some 13C atoms will also be labelled with 2H to ensure narrow lines for enzyme-bound species. To optimize the information obtained from the 13C NMR characterization of enzyme-bound species, several complementary studies will be carried out. The equilibrium between species bound at the active site will be determined using radioactive substrates and acid quench techniques. Equilibrium binding studies will determine the stoichiometry of active site(s). Molecular biological techniques will be used to increase the expression of CMPDH from the cloned tyrA gene. The purification of CMPDH will be optimized. Studies will be carried out to optimize the stability of the enzyme under conditions appropriate for the 13C NMR studies.
|Rajagopalan, J S; Taylor, K M; Jaffe, E K (1993) 13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase. Biochemistry 32:3965-72|