The soluble alpha-glycerophosphate oxidase from Enterococcus casseliflavus. Sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme.
The soluble flavoprotein alpha-glycerophosphate oxidase from Enterococcus casseliflavus catalyzes the oxidation of a "non-activated" secondary alcohol, in contrast to the flavin-dependent alpha-hydroxy- and alpha-amino acid oxidases. Surprisingly, the alpha-glycerophosphate oxidase sequence is 43% identical to that of the membrane-associated alpha-glycerophosphate dehydrogenase from Bacillus subtilis; only low levels of identity (17-22%) result from comparisons with other FAD-dependent oxidases. The recombinant alpha-glycerophosphate oxidase is fully active and stabilizes a flavin N(5)-sulfite adduct, but only small amounts of intermediate flavin semiquinone are observed during reductive titrations. Direct determination of the redox potential for the FAD/FADH2 couple yields a value of -118 mV; the protein environment raises the flavin potential by 100 mV in order to provide for a productive interaction with the reducing substrate. Steady-state kinetic analysis, using the enzyme-monitored turnover method, indicates that a ping-pong mechanism applies and also allows the determination of the corresponding kinetic constants. In addition, stopped-flow studies of the reductive half-reaction provide for the measurement of the dissociation constant for the enzyme. alpha-glycerophosphate complex and the rate constant for reduction of the enzyme flavin. These and other results demonstrate that this enzyme offers a very promising paradigm for examining the protein determinants for flavin reactivity and mechanism in the energy-yielding metabolism of alpha-glycerophosphate.
Parsonage
D
D
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
Luba
J
J
Mallett
T C
TC
Claiborne
A
A
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D004227:Dithionite; D016983:Enterococcus / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D005993:Glycerolphosphate Dehydrogenase