A NEW METHOD FOR DETERMINING RIBOFLAVIN KINASE-ACTIVITY

A new method has been developed for measuring the activity of riboflavin kinase (ATP: riboflavin 5'-phosphotransferase; EC 2.7.1.26), the enzyme, which phosphorylates riboflavin and thereby produces flavin mononucleotide (FMN). When the phosphorylation is completed, the excess of the substrate is selectively transported into the cells of a yeast mutant, Pichia guilliermondii VKPM Y-432, by a highly specific riboflavin permease. Then the cells are removed from the reaction mixture, and the concentration of FMN formed during the reaction is determined directly by measuring the sample fluorescence. The method is simple and highly sensitive (as little as 0.5 mu U enzyme per ml can be detected). Its applications include measurements of riboflavin kinase activity in preparations of different purity, kinetic studies, and determination of kinetic parameters (V, K-m, K-i) for natural substrates, synthetic substrate analogs of vitamin B-2, or inhibitors of the enzyme.