Cobalt(III) labeled aspartokinase-homoserine dehydrogenase of Escherichia coli.
The kinase activity of the threonine-sensitive aspartokinase-homoserine dehydrogenase enzyme complex of Escherichia coli was selectively inactivated by Co(III) incorporation. Incubation of the enzyme with Co(II) in the presence of oxygen or H2O2 resulted in incorporation of one Co(III) per subunit. The cobalt(III) bound to the enzyme was not removable by dialysis and presumably results from formation of "inert" coordination complexes with ligands contributed by the enzyme. Cobalt was released from the enzyme by incubation with dithiothreitol but not by metal chelating agents. The Co(III)-labeled enzyme was aspartokinase inactive but still retained 60% of its original homoserine dehydrogenase activity. Studies of the time course of inactivation showed aspartokinase inactivation paralleled Co(III) incorporation. The residual dehydrogenase activity of aspartokinase inactive enzyme was still inhibited by threonine Thus, Co(III) incorporation seems to result in a specific inactivation of kinase activity which permits enumeration of the number of aspartokinase sites. Limited alpha-chymotrypsin digestion of Co(III)-enzyme produced homoserine dehydrogenase-active fragments devoid of Co(III), further confirming the specificity of the labeling procedure. Aspartokinase inactivation obtained without concomitant desensitization of homoserine dehydrogenase to threonine inhibition suggests that kinase active site integrity is not required for threonine binding and inhibition of homoserine dehydrogenase.
D001225:Aspartokinase Homoserine Dehydrogenase / Q000378:metabolism*; D001665:Binding Sites; D002918:Chymotrypsin; D003035:Cobalt / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding
D009097:Multienzyme Complexes; D003035:Cobalt; D001225:Aspartokinase Homoserine Dehydrogenase; D002918:Chymotrypsin; D008274:Magnesium; D004229:Dithiothreitol