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Purification, properties and mechanistic classification of Aspergillus niger sulfhydryl oxidase

Rebecca Sue de la Motte, University of Nebraska - Lincoln

Abstract

Sulfhydryl oxidases are enzymes which catalyze the conversion of thiol compounds to their corresponding disulfides with concomitant reduction of molecular oxygen to hydrogen peroxide. Such an enzyme was purified from a brine extract of Aspergillus niger mycelia by a method which involved methanol fractionation in the brine, acetone fractionation in citrate buffer at pH 4.0, hydroxylapatite chromatography in acetone/citrate medium, and DEAE cellulose chromatography in acetate buffer at pH 5.5. The sulfhydryl oxidase thus isolated had a specific activity of about 75 $\mu$mol O$\sb2$ consumed per mg protein toward the substrate glutathione; showed a pH optimum of about 5.5 for the same substrate; utilized other thiol substrates, including the protein ribonuclease A, but would not utilize pyridine nucleotides as electron acceptors; was a dimer of subunit molecular weight 53 kD; consisted of 23% carbohydrate by weight; possessed three half-cystines and 0.84 FAD molecules per subunit, and exhibited the spectral properties expected of a flavin oxidase. Sulfhydryl oxidase was classified as a flavin disulfide oxidoreductase, i.e. a flavoprotein which possesses a redox-active disulfide in its active site. The enzyme could be alkylated at a single half-cystine residue by haloacetamides in the presence, but not in the absence, of a reducing substrate. A cystine disulfide was cleaved when the enzyme was thus modified. The resulting enzyme inactivation exhibited pseudo-first-order kinetic properties which were consistent with modification at a single residue. Monoalkylation induced a profound spectral alteration which involved loss of the enzyme's flavin absorbance maxima at 364.5 and 442.5 nm, with formation of a single new maximum of subunit molar absorptivity 8650 M$\sp{-1}$ cm$\sp{-1}$ at 391 nm. This spectral form developed in a manner proportional to the extent of enzyme inactivation, and is considered to be indicative of the formation of a C4a adduct between the flavin's isoalloxazine moiety and a thiol produced by cleavage of an active site disulfide during catalytic turnover.

Subject Area

Molecular biology|Biochemistry|Microbiology

Recommended Citation

de la Motte, Rebecca Sue, "Purification, properties and mechanistic classification of Aspergillus niger sulfhydryl oxidase" (1996). ETD collection for University of Nebraska-Lincoln. AAI9620337.
https://digitalcommons.unl.edu/dissertations/AAI9620337

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