A continuous fluorometric assay for flavokinase. Properties of flavokinase from Peptostreptococcus elsdenii.
A continuous fluorometric assay that utilizes apoflavodoxin as a trapping agent for riboflavin 5'-phosphate (FMN) has been developed for flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26). Use of this assay is illustrated in a procedure for the partial purification of flavokinase from the strict anaerobe Peptostreptococcus elsdenii. The purified enzyme catalyzed the formation of 8.3 nmol FMN - min-1 - mg-1 at 37 degrees C and had apparent Km values for riboflavin and ATP of 10 and 4.7 micronM, respectively. ATP could be replaced by ADP (22% of the rate observed with ATP) but not by GTP. The enzyme also phosphorylated 5-deaza- and 8-bromoriboflavin with activities of 15 and 70%, respectively, of that with riboflavin; it was inactive with iso riboflavin and deoxyriboflavin.
D007700:Kinetics; D010462:Peptostreptococcus / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D012256:Riboflavin; D013050:Spectrometry, Fluorescence
D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022532:riboflavin kinase; D012256:Riboflavin