The binding of indole to the alpha-subunit and beta2-subunit and to the alpha2beta2-complex of tryptophan synthase from Escherichia coli. Identification of a second indole-binding site on the alpha-subunit.
The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual alpha and beta2-subunits and to the alpha2beta2-complex of tryptophan synthase was studied by equilibrium dialysis. The use of [14C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the alpha-subunit. The binding of indole to the active site of the alpha-subunit is weak (Kd = 18mM). A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the alpha-subunit. The beta2-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain. The alpha2beta2-complex retains one or two sites per alphabeta-equivalent of relatively high affinity (Kd = 1.2 mM). The active sites of the component alpha and beta-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole. These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007211:Indoles; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding; D014367:Tryptophan Synthase / Q000378:metabolism
D007211:Indoles; D008024:Ligands; D046911:Macromolecular Substances; D014367:Tryptophan Synthase