5-Azidoindole binding to the Escherichia coli tryptophan synthase α2β2 complex

Abstract

The tryptophan synthase α 2 β 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN → Trp activity (0.1 m m), in the absence of serine, the enzyme was increasingly inactivated (up to 70–80%) concomitant with the progressive binding of a net of 2 mol AzIN per αβ equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN → InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP → Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1–0.7 m m), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/αβ equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated β 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.