Refolding of a bifunctional enzyme and its monofunctional fragment.

Abstract

The renaturation of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase [from Escherichia coli] II was studied by using the reappearance of its 2 activities. The same kinetics of renaturation are obtained for the dehydrogenase (EC 1.1.1.3) and the kinase activity (EC 2.7.2.4). The mechanism of refolding of the enzyme apparently involves 2 steps, a folding step occurring within a monomer and a subsequent dimerization step. The reappearance of the 2 activities depends on this dimerization step, suggesting that monomeric species are inactive. A proteolytic fragment possessing full dehydrogenase activity is able to renature, as judged by the recovery of its activity. In this case also, the refolding depends on the formation of dimeric species. The refolding of this fragment is much faster than that of the dehydrogenase region in the intact enzyme. Although the dehydrogenase region can refold by itself when isolated as a fragment, refolding of this same region in the whole protein probably involves interactions with the remainder of the protein.