K. Kiuchi, M. Nishikimi, K. Yagi
Sep 28, 1982
Citations
5
Influential Citations
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Journal
Biochemistry
Abstract
L-Gulonolactone oxidase was purified from chicken kidney to homogeneity by a five-step method with a recovery of ca. 30%. The molecular weight of the purified enzyme under nondenaturing conditions was estimated to be 400 000 by gel filtration on Sepharose CL-6B, while that of the dissociated enzyme was 50 000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The latter value was comparable to the minimum molecular weight based on the content of the enzyme-bound flavin. The absorption spectrum of the enzyme showed maxima at 454, 352, and 278 nm. The spectrum in the visible and near-UV range was changed to that of the reduced form by the addition of a substrate, L-gulono-γ-lactone, under anaerobic conditions, and the reduced form of the enzyme was returned to the oxidized form by aeration. A peptide containing covalently bound flavin was isolated by tryptic-chymotryptic digestion of the enzyme and was shown to release 5’-AMP by the treatment with nucleotide pyrophosphatase, indicating that the flavin moiety is FAD. The structure of (aminoacyl)flavin derived from the peptide was identified to be 8α-(N-histidyl)riboflavin by high-voltage paper electrophoresis. In addition to L-gulono-γ-lactone, this enzyme attacked a number of hexonic acid lactones whose hydroxyl group on C(2) has the same configuration as that of L-gulono-γ-lactone. Stoichiometry of the enzyme reaction was determined as L-gulono-γ-lactone + O2 → L-ascorbic acid + H2O2. With L-gulono-γ-lactone as the substrate, the transient appearance of an intermediate, which is considered to be 2-oxo-L-gulono-γ-lactone, was demonstrated by spectrophotometric tracing of the formation of L-ascorbic acid. The values of Km and molecular activity obtained at low concentrations of substrate were 7 μM and 139 min.