title: Identification of an active dimeric form of aspartase as a denaturation intermediate.
abstract: The guanidine-HCl (Gu-HCl)-induced denaturation of a tetrameric enzyme, aspartase from Escherichia coli has been studied by size-exclusion chromatography, and circular dichroism and fluorescence spectroscopies. The size-exclusion analysis showed that in the presence of 0.4 M Gu-HCl, the enzyme has a dimeric structure with 45% of the native activity. The fluorescence and CD studies showed that only a small change occurred in the secondary and tertiary structures in 0.4 M Gu-HCl. In the range of 0.4 to 1 M Gu-HCl, decrease in the activity was observed as the secondary and tertiary structures were disrupted, whereas the dimeric enzyme did not dissociate into inactive monomer until 1 M Gu-HCl. When the enzyme was denatured in less than 1 M Gu-HCl, more than 90% of the original activity was recovered from the renaturation reaction, indicating that the dissociation process from tetramer to dimer is reversible. In contrast, the renaturation yield was 43% when the enzyme was diluted from more than 1 M Gu-HCl, indicating that the process of dissociation into monomer is not reversible. Thus, we identified an active dimeric form as a denaturation intermediate in this study, although the intermediates (including dimer) that were detected in renaturation experiments at low temperature were inactive, as reported previously [Imaishi, H., Yumoto, N., & Tokushige, M. (1989) Physiol. Chem. Phys. Med. NMR 21, 221-228].
authors.affiliation: Department of Chemistry, Faculty of Science, Kyoto University.
authors.forename: S
authors.initials: S
authors.lastname: Murase
authors.forename: Y
authors.initials: Y
authors.lastname: Kawata
authors.forename: N
authors.initials: N
authors.lastname: Yumoto
mesh_terms: D001220:Aspartate Ammonia-Lyase / Q000737:chemistry*; D001704:Biopolymers; D002850:Chromatography, Gel; D002942:Circular Dichroism; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
chemical_list: D001704:Biopolymers; D001220:Aspartate Ammonia-Lyase