Ligand-binding studies on light riboflavin synthase from Bacillus subtilis.

Abstract

Light riboflavin synthase of Bacillus subtilis is a trimer of identical subunits. The enzyme catalyzes the transfer of a four-carbon moiety from one molecule of 6,7-dimethyl-8-ribityllumazine to a second molecule of this compound. Binding of substrate and product analogues to the enzyme was studied by analytical ultra-centrifugation and fluorescence titration. The ligands used in these experiments inhibit the enzyme activity competitively. Each enzyme subunit was shown to bind two molecules each of various analogues of the enzyme substrate, 6,7-dimethyl-8-ribityllumazine, at nonidentical sites. On the other hand, each subunit binds only one molecule of the product, riboflavin, or 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, an analogue of the second product. The complex of the enzyme with the substrate analogue, 7-methyl-8-ribityllumazine, was studied by absorbance and difference absorbance measurements. The data suggest that binding of the lumazine to the donor site of the enzyme involves a nucleophilic attack at carbon 7 of the lumazine ring with formation of a covalent hydrate or a related structure.