Studies on the Quaternary Structure of the Threonine-Sensitive Aspartokinase-Homoscrine Dehydrogenase of Escherichia coli.

The threonine-sensitive aspartokinase-homoserine dehydrogenase of Escherichia coli can exist either as a tetramer (M_r = 360000) or a dimer, depending on the conditions. The subunit interactions of this enzyme have been investigated by reacting-enzyme sedimentation, gel filtration, and electrophoresis. Mild proteolysis with trypsin converts the native enzyme to a dimer which is smaller (M_r = 110000) than the native dimer (M_r = 175000). The new dimer fragment possesses homoserine dehydrogenase activity, sediments as a 6-S species in reacting-enzyme sedimentation but does not show aspartokinase activity. The dehydrogenase activity is lower, however, and the inhibition by threonine of this reaction has largely been lost. L-Threonine or to a lesser extent L-aspartate plus potassium ion protect aspartokinase-homoserine dehydrogenase against proteolysis probably by stabilization of the tetrameric form of the enzyme. Conditions which promote the formation of dimer (removal of effectors) increase the susceptibility to proteolysis. Various treatments cause dissociation of tetrameric aspartokinase-homoserine dehydrogenase, namely alteration of pH, removal of effectors, proteolysis, or sulfhydryl modification. Singly or in combination these treatments always resulted in the formation of dimers (6 S to 8 S in reactingenzyme sedimentation) rather than monomers. This finding strongly indicates that the directs are formed in all cases by disruption of the same intersubunit bonding domain of the tetramer. Based upon these and other studies, we have proposed a subunit interaction model for aspartokinase-homoserine dehydrogenase. The model describes a molecule in which each of four identical subunits contains somewhat independent entities corresponding to aspartokinase and homoscrine dehydrogenase activities. The native tetramer is held together solely by isologous interactions, two between aspartokinase entities and two between regions in which aspartokinase and homoserine dehydrogenase entities overlap. Dissociation by all mild treatments is explained by cleavage of the isologous interaction between aspartokinase entities resulting in loss of aspartokinase activity and production of a homoserine dehydrogenase dimeric unit. [ABSTRACT FROM AUTHOR]