Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I

Abstract

In the range of guanidine hydrochloride concentrations from 0.2-1.2 M, aspartokinase-homoserine dehydrogenase I loses its enzymatic properties, both kinase and dehydrogenase activities and their allosteric inhibition by L-threonine. Ligands which stabilize the tetrameric native structure protect the enzyme against inactivation. Under some conditions, all the functional properties do not disappear at the same rate; an intermediate species possessing only the kinase activity can be detected. Several arguments suggest that this partly active intermediate has a monomeric structure. Deactivation of aspartokinase-homoserin dehydrogenase I is a stepwise process, compatible with the reverse of the previously described reactivation. The same measurements performed with a monofunctional fragment carrying the dehydrogenase activity show that the loss of dehydrogenase activity is the same whether or not the polypeptide chain is intact or lacks the kinase region; this finding suggests that the protein is composed of independent regions. The influence of protein aggregation in studying unfolding-refolding of oligomeric enzymes is also discussed.