5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex.
The tryptophan synthase alpha 2 beta 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 mM), 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 alpha beta 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 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta 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 beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.
D001386:Azides / Q000378:metabolism* / Q000528:radiation effects; D055598:Chemical Phenomena; D002621:Chemistry; D003624:Darkness; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D012694:Serine / Q000494:pharmacology; D013379:Substrate Specificity; D014367:Tryptophan Synthase / Q000378:metabolism* / Q000528:radiation effects
D001386:Azides; D010446:Peptide Fragments; D012694:Serine; C037179:5-azidoindole; D014367:Tryptophan Synthase