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 cr-glycerophosphate oxidase fi om Enterococcu s casseliflavus catalyzes the oxidation of a ''non-activated'' seconda ry alcohol, in contrast to the flavin-dependent alpha-hydroxy- and cy- amino acid oxidases. Surprisingly, the cr-glycerophosphate oxidase seq uence is 43% identical to that of the membrane-associated cu-glyceroph osphate dehydrogenase from Bacillus subtilis; only low levels of ident ity (17-22%) result hom comparisons with other FAD-dependent oxidases. The recombinant alpha-glycerophosphate oxidase is fully active and st abilizes a flavin N(ti)-sulfite adduct, but only small amounts of inte rmediate flavin semiquinone are observed during reductive titrations. Direct determination of the redox potential for the FAD/FADH(2) 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 w ith the reducing substrate, Steady-state kinetic analysis, using the e nzyme-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-re action provide for the measurement of the dissociation constant for th e enzyme alpha-glycerophosphate complex and the rate constant for redu ction of the enzyme flavin. These and other results demonstrate that t his enzyme offers a very promising paradigm for examining the protein determinants for flavin reactivity and mechanism in the energy-yieldin g metabolism of alpha-glycerophosphate.