Allosteric linkages between beta-site covalent transformations and alpha-site activation and deactivation in the tryptophan synthase bienzyme complex.

This work examines two aspects of the catalytic mechanism and allosteric regulation of the tryptophan synthase bienzyme complex from Salmonella typhimurium: (a) the chemical mechanism by which indole and other nucleophiles react with the enzyme-bound alpha-aminoacrylate Schiff base intermediate, E(A-A), to form quinonoidal intermediates, E(Q), and (b) the effects of covalent transformations at the beta-site on the catalytic activity of the alpha-site. Transient kinetic studies in combination with alpha-secondary deuterium isotope effects are undertaken to determine the mechanism of nucleophile addition to E(A-A). These studies establish that nucleophilic attack is best described by a two-step reaction sequence consisting of a binding step that is followed by Michael addition to the conjugated double bond of E(A-A). Analysis of isotope effects suggests that the transition state for indole addition gives an E(A-A) beta-carbon that resembles an sp3 center, while the stronger nucleophiles, indoline and beta-mercaptoethanol, have transition states that appear to more closely resemble an sp2 beta-carbon. The effects of beta-site covalent transformations on alpha-site catalysis were studied using quasi-stable beta-site intermediates and the alpha-site substrate analogue 3-[6-nitroindole]-D-glycerol 3'-phosphate (6-nitro-IGP). It was found that the cleavage of 6-nitro-IGP is strongly activated by the formation of E(A-A) and various E(Q) species at the beta-site but not by external aldimine species. Therefore, we conclude that the conversion of the L-Ser external aldimine to E(A-A) is the beta-site process which activates the alpha-site, while conversion of E(Q) to the L-Trp external aldimine triggers deactivation of the alpha-site. These findings are discussed within the context of allosteric regulation of substrate channeling in tryptophan synthase catalysis.