title: Studies on the structure and mechanism of Streptococcus faecium L-alpha-glycerophosphate oxidase.
abstract: An FAD-containing L-alpha-glycerophosphate oxidase has been purified to homogeneity from Streptococcus faecium. The purified protein exists as a dimer (subunit Mr = 65,000); each subunit contains 1 mol of FAD. The enzyme contains no iron, as determined by atomic absorption spectroscopy. The alpha-glycerophosphate oxidase reacts reversibly with sulfite to form a covalent N(5) adduct; it preferentially binds the anionic form of the native oxidized FAD, and it also stabilizes the p-quinonoid form of 8-mercapto-FAD. The enzyme shows an unusually high reactivity with ferricyanide in the absence of oxygen; however, there is no evidence for any superoxide ion (O2-.) generation under standard assay conditions. Dithionite titrations of the enzyme reveal an unusual pH dependence for the stabilization of the flavin semiquinone; only at pH 8.5 does significant anionic semiquinone accumulate. L-alpha-Glycerophosphate rapidly reduces the enzyme-bound FAD; in addition, a small amount of catalytically insignificant red semiquinone appears under these conditions. The 5-deaza-FAD-reconstituted enzyme is also reduced by substrate, strongly suggesting that a radical mechanism is not involved in the oxidation of alpha-glycerophosphate. Furthermore, nitroethane anion reduces the native enzyme; this observation suggests that an electron transfer mechanism involving a substrate carbanion is possible with this enzyme.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D004227:Dithionite / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013053:Spectrophotometry; D013291:Streptococcus / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D005993:Glycerolphosphate Dehydrogenase