ALLOSTERIC INTERACTIONS COORDINATE CATALYTIC ACTIVITY BETWEEN SUCCESSIVE METABOLIC ENZYMES IN THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX

Tryptophan synthase from enteric bacteria is an alpha-2-beta-2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha-beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha-2-beta-2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. Beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site. These results show that ligand-dependent allosteric interactions between heterologous subunits in the bienzyme complex serve to coordinate catalytic events at the alpha- and beta-active sites to ensure the efficient synthesis of L-Trp. We propose that these ligand-dependent allosteric phenomena are accompanied by conformational transitions in both the alpha- and beta-subunits between "open" and "closed" conformations that control ligand affinity and catalytic activity.