The enzymatic phosphorylation of riboflavin.

While the existence and coenzymatic function of riboflavin-5-phosphate’ have been recognized for some time, the meansby which this substance is synthesized from the free vitamin have not been satisfactorily demonstrated. Verzar and coworkers (3,4) have reported extensive phosphorylation of riboflavin by means of inorganic phosphate in preparations of intestinal mucosa. The synthesis of FMN by such reversal of phosphatase action seemed an unlikely biological mechanism in view of the energetics of the reaction, but we none the less attempted to repeat this finding, closely duplicating their experimental conditions. Although these workers had reported as high as 50 per cent conversion of riboflavin to FMN, we were not able to demonstrate any FMN formation. Attempts to phosphorylate riboflavin with ATP in minces, homogenates, extracts, and acetone powders of hog and rat intestinal mucosa were likewise unsuccessful; added FMN was so rapidly split by phosphatase action, even in the presence of inhibitors such as fluoride or cysteine (5), that any formation of FMN could not have been detected. It was possible, however, to demonstrate phosphorylation of riboflavin in suspensions of dried brewers’ yeast in which interfering enzymes could be inhibited by the addition of fluoride. The present paper describes the isolation and properties of the enzyme which catalyzes this transphosphorylation. On the basis of the specificity and stoichiometry of the reaction, the name “flavokinase” is suggested for the enzyme.