Riboflavin 5'-pyrophosphate: a contaminant of commercial FAD, a coenzyme for FAD-dependent oxidases, and an inhibitor of FAD synthetase.
Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5'-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apoprotein of porcine D-amino-acid oxidase, values determined for RPP were 8.4 microM for Km and 0.10 for Vmax compared to 0.47 microM and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 nM for Km and 0.02 for Vmax and 45 nM and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a Ki value for RPP against FMN was determined to be 9 microM where Km for FMN was 5.5 microM. These studies illustrate the value of riboflavin 5'-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.
Department of Biochemistry, Rollins Research Center, Emory University, Atlanta, Georgia 30322.
H A
HA
Hartman
D E
DE
Edmondson
D B
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McCormick
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003067:Coenzymes / Q000494:pharmacology*; D003605:D-Amino-Acid Oxidase / Q000502:physiology; D004340:Drug Contamination; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis* / Q000737:chemistry; D005415:Flavins / Q000032:analysis; D005949:Glucose Oxidase / Q000502:physiology; D009682:Magnetic Resonance Spectroscopy; D009713:Nucleotidyltransferases / Q000032:analysis / Q000037:antagonists & inhibitors*
D003067:Coenzymes; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; C076415:riboflavin 5'-pyrophosphate; D005486:Flavin Mononucleotide; D005949:Glucose Oxidase; D003605:D-Amino-Acid Oxidase; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase