Lumazine synthase catalyzes the condensation of 5-amino-6-(D- ribitylamino)-2,4- (1H,3H)pyrimidinedione with the novel carbohydrate, L- 3,4-dihydroxy-2-butanone-4-phosphate to form the immediate precursor of riboflavin, 6,7-dimethyl-8-(D-ribitylamino)lumazine. Recent developments in our understanding of lumazine synthase include the identification of L- 3,4.dihydroxy-2-butanone-4-phosphate as one of the substrates and the 2.4 A X-ray structure of reconstituted (beta-60 capsids of lumazine synthase complexed with phosphate and the substrate analog, 5-nitro-(D- ribitylamino)-2,4-(lH,3H)pyrimidinedione. This new information has opened up new possibilities for the study of the structure and mechanism of lumazine synthase. The identification of the carbohydrate precursor has allowed a mechanistic hypothesis to be advanced concerning the pathway for the conversion of the substrates into the lumazine product. In addition, the X-ray structure has made it possible to propose a mechanism for how the enzyme functions in catalyzing the reaction and a hypothetical binding geometry for the substrates and proposed reaction intermediates. In order to test these new ideas, metabolically stable substrate and reaction intermediate analogs are required. The present research project involves the design and synthesis of a set of lumazine synthase probes that will be valuable in obtaining evidence about the mechanism of the reaction catalyzed by the enzyme. The metabolically stable ligands have been designed to represent certain hypothetical intermediates in the proposed mechanism. After synthesis, they will be bound to the active site of lumazine synthase by ligand driven aggregation or by diffusion and the X- ray structures of the resulting complexes will be determined. In addition, the structures of the complexes will be investigated by REDOR and TENDOR solid state NMR techniques. REDOR NMR offers the advantages of 0.1 resolution and applicability to non-crystalline protein samples, and is therefore complementary to X-ray techniques. The recent identification of L-3,4-dihydroxy-2-butanone-4-phosphate as a substrate of lumazine synthase has also made it possible, for the first time, to determine the standard thermodynamic and kinetic parameters of lumazine synthase inhibitors. The inhibition constants (Ki) and dissociation constants (KD) of the new inhibitors will be determined, arid ligand displacement studies will also be performed. The binding stoichiometry will be determined. These experiments will be performed on both heavy riboflavin synthase as well as on hollow beta-60 capsids obtained by ligand-driven aggregation. Besides variation of the ligands studied, all potential mutations of the protein can be made by site-directed mutagenesis, and the recombinant proteins can be expressed in E. coil. The mechanistic interpretation will be based on protein as well as ligand modification. Uniformly 15N-labeled protein and fluorine-containing protein will be especially useful in the intepretation of the REDOR spectra.