Biosynthesis of Ascorbate in Yeast

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Abstract
An enzyme from S. cerevisiae which catalyzes the reaction: L-galactonolactone + O2 .fwdarw. L-ascorbate + H2O2 was purified 466-fold from the mitochondria fraction of a yeast homogenate. The enzyme has several properties that are different from the L-galactonolactone oxidase described by Nishikimi et al. By gel filtration in the presence of sodium deoxycholate, an apparent MW of 70,000 was obtained for the active enzyme. Polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave a MW of 74,000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis [SDS-PAGE] showed only 1 protein band corresponding to a MW of 18,000. A tetrameric structure of the enzyme is thereby suggested. The substrate specificity is confined to the aldonoacid lactones L-galactono-, D-altrono-, L-fucono-, D-arabino and D-threono-1,4-lactones. Competitive inhibition was demonstrated with L-gulono- and D-galactono-1,4-lactones. p-Chloromercuriphenyl sulfonate, iodoacetamide, N-ethylmaleimide, sulfite and sulfide were all inhibitory to the enzyme. No effect was seen when cyanide, azide, EDTA, .alpha.,.alpha.''-bipyridyl or bathocuproine disulfonate was added. An apparent Km of 0.3 mM with L-galactonolactone as a substrate occurred. The Km for O2 was 0.18 mM. The pH/activity curve exhibited a maximum around pH 8.9 and a shoulder at pH 6.5. Evidence of a covalently bound flavin coenzyme and involvement of an Fe-S cluster was obtained from difference spectra of oxidized minus substrate-reduced enzyme with peaks or shoulders of the oxidized enzyme at 475, 445, 410, 375 and 350 nm. With SDS-PAGE the enzyme subunit(s) had a bright yellow fluorescence after fixation in 7% acetic acid or 5% formaldehyde. The galactonolactone oxidase is stable with 50% activity being lost in 6 mo. at +5.degree. C.