The Mechanism of Tryptophan Binding to Tryptophan Synthase from Escherichia coli

Abstract

The kinetics of the binding of L-tryptophan to the .alpha.2-holo-.beta.2 complex of tryptophan synthase (EC 4.2.1.20) from E. coli were measured by rapid-mixing under conditions where tryptophan release is mainly rate-determining in tryptophan synthesis. The dependence of the 3 observable rate processes on the concentration of L-tryptophan suggests a mechanism in which a rapid binding step is followed by 2 isomerizations. The effect of the substrate analog 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 2 alternative modes of binding of L-tryptophan exist on the enzyme. The effects of protons, indole and indolepropanol phosphate on the 3 rate processes explain the dependence of kcat on the 3 non-competitive ligands.