The Binding of Indole to the α‐Subunit and β2‐Subunit and to the α2β2‐Complex of Tryptophan Synthase from Escherichia coli

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Abstract
The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual α and β2‐subunits and to the α2β2‐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 α‐subunit. The binding of indole to the active site of the α‐subunit is weak (Kd= 18 mM). 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 α‐subunit. The β2‐subunit binds indole only weakly (Kd= 12 mM) to many (n = 10) sites per polypeptide chain. The α2β2‐complex retains one or two sites per αβ‐equivalent of relatively high affinity (Kd= 1.2 mM). The active sites of the component α and β‐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.