A study of allosteric mechanisms involved in the regulation of substrate channeling in tryptophan synthase.

摘要

The tryptophan synthase bienzyme complex is used to study allosteric communication and substrate channeling between subunits. The alpha- and beta-sites are connected by a 25-A-long tunnel through which the substrate indole, cleaved from 3-indole D-glycerol 3'-phosphate (IGP) in the alpha-reaction, is channeled for reaction at the beta-site with L-Serine. In this work, the novel amino acid dihydroiso-L-tryptophan (DIT) was used as an analogue of L-Trp. When the alpha-site substrate analogue alpha-D,L-glycerol phosphate (GP) is bound, the DIT reaction was found to give a rapid formation and dissipation of indoline quinonoid species, E(Q)Indoline, followed by a very slow reappearance of E(Q)Indoline. With GP bound to the alpha-site, the indoline generated by DIT cleavage in the first turnover is trapped within the enzyme complex. To help study the allosteric effects of ligands bound to the alpha-site, a series of fluorine containing substrate analogs were developed. The compound N-(4-trifluoromethoxybenzoyl)-2-amino-l-ethylphosphate (F6) was characterized and its effects on different reactions of the tryptophan synthase enzyme are presented herein. The indoline leakage mechanism in the DIT reaction was further studied and the escape was found to most likely be through the alpha-site while the alpha-site ligand (ASL) was temporarily disengaged. It was also discovered that the ASL glycerol phosphate (GP) had synergistic binding effects when used with the indole analog benzimidazole (BZI) which produced much stronger indoline trapping effects during the DIT reaction. Lastly, it was found that the natural alpha-site substrate IGP produces the strongest indoline trapping effect in the DIT reaction, and thus IGP must be the most effective allosteric ASL. The binding of IGP to the alpha-site also had the effect of slowing DIT entry into the beta-site, indicating that IGP binding can close or partially close the beta-site even in the absence of L-Ser.