On the mechanism of action of Escherichia coli tryptophan synthase Steady-state investigations

Abstract

Tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole), EC 4.2.1.20) synthesizes l-tryptophan from indoleglycerol phosphate and l-serine, releasing glyceraldehyde 3-phosphate, or from indole and l-serine. The latter reaction (B reaction), catalyzed either by the β 2 species or by the (α 2β 2) complex, has been studied by steady-state methods. A sequential mechanism is indicated. Inhibition experiments with the substrate analogue benzimidazole were carried out in order to distinguish between random and ordered mechanisms. The results are compatible with a random sequential mechanism. The dissociation constants of the enzyme-substrate complexes are evaluated. When catalyzed by the tetrameric complex (α 2β 2) the B reaction is inhibited by higher concentrations of the substrate indole. This inhibition does not follow the usual substrate inhibition pattern. The question whether the binding of indole to the α-subunit exerts an inhibitory effecton the β 2 species, possibly by reversing the activation by the α subunit of the β 2 species, is discussed.