Selective cleavage and modification of the intersubunit disulfide bonds of bovine dopamine beta-monooxygenase: conversion of tetramer to active dimer.
Bovine dopamine beta-monooxygenase 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 degrees C under nondenaturing conditions. A 15-min incubation with 0.5 mM DTT selectively cleaved half of the intersubunit disulfide 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 modified 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.
Ishida
T
T
Department of Biochemistry, Shiga University of Medical Science.
Narita
M
M
Nozaki
M
M
Horiike
K
K
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol; D004299:Dopamine beta-Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D013439:Sulfhydryl Reagents
D004220:Disulfides; D013439:Sulfhydryl Reagents; D004299:Dopamine beta-Hydroxylase; D004229:Dithiothreitol