| Description |
Riboflavin synthetase has been purified approximately 2,500-fold from extracts of dried backer's years. The purified protein exhibited a sedimentation constant of 4.3 in the analytical ultracentrifuge and the molecular weight has been estimated to be 70,000 to 80,000. The apparent Michaelis constant, Km, and the turnover number for the enzyme were estimated to be 1.0 x 10(-5)M and 12 moles riboflavin/mole enzyme/minute at 37?, respectively. A kinetic analysis of the reaction was found to be consistent with a mechanism involving two non-equivalent sites, one binding the 6,7-dimethyl-8-ribityllumazine molecule serving as the donor of the four-carbon moiety transferred in the riboflavin synthetase reaction, the other site binding the lumazine that accept the four-carbon fragment. This reaction model was supported by studies showing that stable, stoichiometric complexes of enzyme and substrate, or of enzyme and inhibitor, could be isolated by Sephadex chromatography. These complexes were estimated to contain one mole of bound material per mole of enzyme, and in the case of 6,7-dimethyl-8-ribityllumazine and 6,7-dimethyl-8-(5'-deoxyribityl)lumazine it was demonstrated that binding occurred exclusively at the donor site of enzyme. Fluorescent polarization experiments have provided evidence for the existence of a ternary complex between the enzyme and substrate and have allowed tentative evaluation of several of the dissociation constants for the enzyme-lumazine complexes. A reaction intermediate different from both 6, 6-dimethyl-8-ribityllumazine and riboflavin could not be demonstrated spectrally or chemically. An intermediate with only a transient existence could not be excluded by the data presented. The addition of glyoxal to incubation mixtures containing enzyme and 6,7-dimethyl-8-ribityllumazine led to the formation of 8-ribityl-lumazine in amounts equivalent to the enzymically formed riboflavin. These results indicate that 4-ribitylamino-5amino-2, 6-dihydrosy-pyrimidine is the second reaction product found in the riboflavin synthetase reaction. |
