Biosynthesis of Ascorbate in Yeast

An enzyme from Saccharomyces cerevisiae which catalyzes the reaction: l ‐galactonolactone + O 2 → l ‐ascorbate + H 2 O 2 has been purified 466‐fold from the mitochondrial fraction of a yeast homogenate. The enzyme has several properties that are different from the l ‐galactonolactone oxidase described by Nishikimi et al. [ Arch. Biochem. Biophys. 191 , 479–486 (1978)]. By gel filtration in the presence of sodium deoxycholate an apparent M r of 70000 was obtained for the active enzyme. Polyacrylamide‐gradient gel electrophoresis in the presence of deoxycholate gave an M r of 74000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis showed only one protein band corresponding to an M r of 18000. 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, α,α'‐bipyridyl or bathocuproine disulfonate was added. An apparent K m of 0.3 mM with l ‐galactonolactone as a substrate was found. The K m for oxygen 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 iron‐sulfur 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. In sodium dodecylsulfate/polyacrylamide gels 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 months at + 5°C.