beta-Site covalent reactions trigger transitions between open and closed conformations of the tryptophan synthase bienzyme complex

Abstract

The tryptophan synthase bienzyme complex (alpha2beta2) from Salmonella tryphimurium catalyzes the final steps in the biosynthesis of L-Trp. To investigate the roles played by conformational change in tryptopthan synthase catalysis, the fluorophore 8-anilino-1-naphthalensulfonate (ANS) is used to identify conformational states. The binding of ANS to the alpha2beta2 bienzyme complex is accompanied by a dramatic enhancement of ANS fluorescence and a shift of the emission maximum from 520 to 482 nm. The ANS binding isotherm is biphasic and consists of a class of moderately high-affinity, noninteracting sites with a stoichiometry of 1 site/alpha beta dimeric unit (Kd' = 62 + or - 15 micrometer) and a much weaker set of non-specific interactions with K'd>1mM. Our findings show that the affinity of the enzyme for ANS is strongly decreased (> 10-fold) by interactions at two loci 30 angstroms apart: (i) the binding of the alpha-site ligands, 3-indole-D-glycerol 3'-phosphate or alpha-glycerol phosphate (GP) or (ii) reaction at the beta-subunit to form either the alpha-aminoacrylate Schiff base, E(A-A), or quinonoid species, E(Q). In contrast, formation of the L-Ser and L-Trp external aldimines E(Aex1) and E(Aex2) at the beta-site causes a 2-3 fold decrease in the affinity of the enzyme for ANS. The combination of E(A-A)or E(Q) with GP brings about almost complete displacement of ANS, indicating that these interactions drive a conformation change in alphabeta subunit pairs which prevents the binding of ANS. These results are consistent with a model which postulates that alphabeta subunit pairs undergo ligand-mediated transitions between open and closed conformations during the catalytic cycle. Consistent with the kinetic data showing that binding of alpha-site ligands increases the affinity of the beta site for L-Ser and that formation of E(A-A) activates the alpha reaction [Brzović, P. S., Ngo, K., & Dunn, M. F. (1992) Biochemistry 31, 3831-3839], while mutations in alpha subunit loops 2 and 6 prevent the ligand- mediated transition to a closed structure [Brzović, P.S., Hyde, C.C., Miles, E.W., & Dunn, M.F. (1993) Biochemistry 32, 10404-10413], we conclude that reciprocal ligand-mediated allosteric interactions between the heterologous subunits promote conformational transitions between open and closed structures in alphabeta subunit pairs which function to coordinate catalytic activities and facilitate the channeling of indole between the two catalytic sites.