title: Allosteric regulation of substrate channeling in tryptophan synthase: modulation of the L-serine reaction in stage I of the beta-reaction by alpha-site ligands.
abstract: In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 A long tunnel. Within the beta-site, indole and l-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give l-Trp. In stage I, l-Ser forms an external aldimine, E(Aex1), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site >30-fold. In stage II, indole reacts with E(A-A) to give l-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for l-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, d-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex1) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex1) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713-7727].
authors.affiliation: Department of Biochemistry, University of California, Riverside, California 92521, USA.
authors.forename: Huu
authors.initials: H
authors.lastname: Ngo
authors.forename: Novelle
authors.initials: N
authors.lastname: Kimmich
authors.forename: Rodney
authors.initials: R
authors.lastname: Harris
authors.forename: Dimitri
authors.initials: D
authors.lastname: Niks
authors.forename: Lars
authors.initials: L
authors.lastname: Blumenstein
authors.forename: Victor
authors.initials: V
authors.lastname: Kulik
authors.forename: Thomas Reinier
authors.initials: TR
authors.lastname: Barends
authors.forename: Ilme
authors.initials: I
authors.lastname: Schlichting
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology*; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D012486:Salmonella typhimurium / Q000201:enzymology*; D012694:Serine / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008024:Ligands; D012694:Serine; D014367:Tryptophan Synthase