STUDIES ON THE SUBUNITS OF ESCHERICHIA-COLI COENZYME-A TRANSFERASE - RECONSTITUTION OF AN ACTIVE ENZYME

The α and β subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified β subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of α subunit. The presence of the substrate, acetoacetyl-CoA, is required to recover the catalytic activity of the β subunit and of mixtures containing purified α and β subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native α2β2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated α subunit. The results indicate that the α subunit serves as a structural subunit, or possibly a maturation subunit, imposing a conformation on the β subunit that is catalytically more competent. © 1979.