Sequential folding of a bifunctional allosteric protein.

Aspartokinase I-homoserine dehydrogenase I (EC 2.7.2.4 and EC 1.1.1.3) a bifunctional and allosteric enzyme, has been renatured from its unfolded and separated polypeptide chains. Folding was measured by the reappearance of each of the two enzymatic activities, kinase and dehydrogenase, and of their allosteric inhibition by the same effector, threonine. The various observed properties yield different kinetics of folding, which shows the presence of intermediates having only some of the functional features of the native enzyme. Apparently, three successive steps can be detected during the folding of aspartokinase I-homoserine dehydrogenase I: first, a monomolecular step leads to a monomeric species with the kinase activity; then an association step leads to a dimeric species with the kinase and dehydrogenase activities, and a threonine-sensitive dehydrogenase; finally, a second association step leads to a tetrameric species with the two activities, both sensitive to threonine. The folding of this large protein appears as a sequential process during which the functional properties are regained successively, as the protein structure becomes more complex. During this process, the two regions of each polypeptide chain respectively responsible for the kinase and dehydrogenase activities seem to acquire their native conformation rather independently of each other.