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- Title
Selective Cleavage and Modification of the Intersubunit Disulfide Bonds of Bovine Dopamine β-Monooxygenase: Conversion of Tetramer to Active Dimer.
- Authors
Ishida, Tetsuo; Narita, Mitauhiro; Nozaki, Mitsuhiro; Horiike, Kihachiro
- Abstract
Bovine dopamine β-monaooxygenase is a tetramer consisting of two disulfide-linked dimers. To examine the role(s) of the intersubunit disulfide bonds in the protein structure and activity, the enzyme was treated with DTT at pH 7.5 and 25°C under nondenaturing conditions. A 15-min incubation with 0.5 mM DTT selectively cleaved half of the intersubunit disulflde bonds. The cleavage did not affect the activity or tetrameric structure of the enzyme. Upon chemical modification of the reduced cysteine residues with 0.1 M iodoacetamide (IAA) for 60 min, half of the tetramer was converted to a dimeric species. The resulting dimeric and tetrameric species exhibited similar kinetic properties, and the Vmax values were decreased by 30% compared to that of the native enzyme. Upon treatment with IAA alone, no dimer species was detected but the enzyme lost 30% of the original activity. Cys514 and Cys516 were selectively modifled by the treatment with DTT and IAA. From these results, we concluded that: (i) chemical modification of the intermolecular disulfide bonds strongly destabilizes the intersubunit interaction; (ii) breakage of the intersubunit interaction does not affect the activity. The reduction mechanism of the intersubunit disulfide bonds and the roles of the intersubunit interactions are discussed.
- Subjects
DOPAMINE; DISULFIDES; MONOOXYGENASES; DIMERS; PROTEINS
- Publication
Journal of Biochemistry, 1996, Vol 120, Issue 2, p346
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a021419