Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the transition to the denatured state with residual structure

Gabriel Zoldák, Anton Zubrik, Andrej Musatov, Marek Stupák, Erik Sedlák

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108 Scopus citations

Abstract

Glucose oxidase (GOX; β-D-glucose:oxygen oxidoreductase) from Aspergillus niger is a dimeric flavoprotein with a molecular mass of 80 kDa/monomer. Thermal denaturation of glucose oxidase has been studied by absorbance, circular dichroism spectroscopy, viscosimetry, and differential scanning calorimetry. Thermal transition of this homodimeric enzyme is irreversible and, surprisingly, independent of GOX concentration (0.2-5.1 mg/ml). It has an apparent transition temperature of 55.8 ± 1.2°C and an activation energy of ∼280 kJ/mol, calculated from the Lumry-Eyring model. The thermally denatured state of GOX after recooling has the following characteristics. (i) It retains ∼70% of the native secondary structure ellipticity; (ii) it has a relatively low intrinsic viscosity, 7.5 ml/g; (iii) it binds ANS; (iv) it has a low Stern-Volmer constant of tryptophan quenching; and (v) it forms defined oligomeric (dimers, trimers, tetramers) structures. It is significantly different from chemically denatured (6.67 M GdmHCl) GOX. Both the thermal and the chemical denaturation of GOX cause dissociation of the flavin cofactor; however, only the chemical denaturation is accompanied by dissociation of the homodimeric GOX into monomers. The transition temperature is independent of the protein concentration, and the properties of the thermally denatured protein indicate that thermally denatured GOX is a compact structure, a form of molten globule-like apoenzyme. GOX is thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.

Original languageEnglish (US)
Pages (from-to)47601-47609
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number46
DOIs
StatePublished - Nov 12 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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