Interaction of tetraiodofluorescein with aspartate transcarbamylase and its isolated catalytic and regulatory subunits.
The interaction of the dye tetraiodofluorescein with native aspartate transcarbamylase and its isolated subunits has been investigated by both binding and activity measurements at 4 and 23 degrees. At room temperature low concentrations of tetraiodofluorescein activate the native enzyme, but high concentrations inhibit the enzyme's activity. At the low temperature the native enzyme is inhibited by all concentrations of dye. Isolated catalytic subunit is very effectively inhibited at both temperatures. For the native enzyme these results are explained by 18 tetraiodofluorescein sites of approximately equal affinity (K = 7.3 X 10(-5) M) on each enzyme hexamer: one class of six sites at the nucleoside triphosphate effector binding sites is responsible for the activation, a second class which competes with the substrate carbamylphosphate causes the inhibition, and a third class does not interact with either the effectors or the substrates. Measurements of tetraiodofluorescein binding to isolated regulatory, catalytic, and p-hydroxymercuribenzoate-inactivated catalytic subunits support the above assignments. This scheme of tetraiodofluorescein binding sites successfully predicts the changes in the tetraiodofluorescein-aspartate transcarbamylase difference spectrum induced by the addition of various ligands. The activity changes induced by the dye are explained if the binding of a single tetraiodofluorescein molecule to one of the six regulatory sites activates all six of the catalytic sites, while while a dye molecule binding to any one of the catalytic sites inactivates only that catalytic site.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D003201:Computers; D004926:Escherichia coli / Q000201:enzymology; D005452:Fluoresceins; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding
D005452:Fluoresceins; D046911:Macromolecular Substances; D001221:Aspartate Carbamoyltransferase