Sequential folding of a bifunctional allosteric protein.

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Abstract
Aspartokinase I-homoserine dehydrogenase I (EC 2.7.2.4 and EC 1.1.1.3), a bifunctional and allosteric enzyme [from Escherichia coli], was renatured from its unfolded and separated polypeptide chains. Folding was measured by the reappearance of each of the 2 enzymatic activies, 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, 3 successive steps can be detected during the folding of aspartokinase I-homoserine dehydrogenase I: 1st, a monomolecular step leads to 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 2nd association step leads to a tetrameric species with the 2 activities, 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 2 regions of each polypeptide chain, respectively, responsible for the kinase and dehydrogenase activities seem to acquire their native conformation rather independently of each other.