The mechanism of tryptophan binding to tryptophan synthase from Escherichia coli.

The kinetics of the binding of L-tryptophan to the alpha 2 holo beta 2 complex of tryptophan synthase from Escherichia coli have been measured by rapid-mixing techniques under conditions where tryptophan release is mainly rate-determining in tryptophan synthesis. The dependence of the three observable rate processes on the concentration of L-tryptophan suggests a mechanism in which a rapid binding step is followed by two isomerizations. The effect of the substrate analogue indolepropanol phosphate on the kinetics of binding and synthesis from L-serine and indole supports a branched mechanism with an unproductive enzyme-ligand complex being the major species. The productive enzyme-ligand complex absorbs light at 473 nm but not at 500 nm. These observations, and binding studies with D-tryptophan, suggest that at least two alterative modes of binding of L-tryptophan exist on the enzyme. The effects of protons, indole and indolepropanol phosphate on the three rate processes explain the dependence of kcat on the three non-competitive ligands.