The tryptophan synthase bienzyme complex is the most extensively documented example of substrate channeling in which the oligomeric unit has been described at near atomic resolution. Transfer of the common metabolite, indole, between the alpha- and the beta-sites occurs by diffusion along a 25-A-long interconnecting tunnel within each alphabeta-dimeric unit of the alpha(2)beta(2) oligomer. The control of metabolite transfer involves allosteric interactions that trigger the switching of alphabeta-dimeric units between open and closed conformations and between catalytic states of low and high activity. This allosteric signaling is triggered by covalent transformations at the beta-site and ligand binding to the alpha-site. The signals are transmitted between sites via a scaffolding of structural elements that includes a monovalent cation (MVC) binding site and salt bridging interactions of betaLys 167 with betaAsp 305 or alphaAsp 56. Through the combined strategies of site-directed mutations of these amino acid residues and cation substitutions at the MVC site, this work examines the interrelationship of the MVC site and the alternative salt bridges formed between Lys beta167 with Asp beta305 or Asp alpha56 to the regulation of channeling. These experiments show that both the binding of a MVC and the formation of the Lys beta167-Asp alpha56 salt bridge are important to the transmission of allosteric signals between the sites, whereas, the salt bridge between betaK167 and betaD305 appears to be only of minor significance to catalysis and allosteric regulation. The mechanistic implications of these findings both for substrate channeling and for catalysis are discussed.
In the absence of other substrates, L-Ser reacts rapidly with the tryptophan synthase alpha 2 beta 2 bienzyme from Salmonella typhimurium at pH 7.8 and 25 degrees C to give an equilibrating mixture of species dominated by comparable amounts of the L-Ser external aldimine Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base, E(A-A). The D-isomer of Ser is unreactive toward alpha 2 beta 2, and therefore, D,L-Ser can be used in place of L-Ser for investigations of catalytic mechanism. Due to the equilibrium isotope effect, when alpha-2H-D,L-Ser is substituted for alpha-1H-D,L-Ser, the position of equilibrium is shifted in favor of E(Aex1). On a much slower time scale, the 2H sample undergoes the exchange of enzyme bound 2H for the 1H of solvent water and is converted to a distribution of E(Aex1) and E(A-A) identical to that obtained with the 1H sample. This slow exchange indicates that the proton abstracted from the alpha-carbon of E(Aex1) is sequestered within a solvent-excluded site in E(A-A). Analysis of the UV/vis spectra gave an isotope effect on the equilibrium distribution of E(Aex1) and E(A-A) of KH/KD = 1.80 +/- 0.18. This large equilibrium isotope effect is the consequence of an unusual isotope fractionation factor of 0.62 for the residue which functions as the base to deprotonate and protonate the alpha-carbon proton in E(Aex1). A fractionation factor of 0.62 qualifies as evidence for the involvement of a low-barrier H-bond (LBHB) in this equilibration. Since this effect arises from abstraction of the alpha-proton from E(Aex1), the LBHB must be associated with the E(A-A) species. In contrast to weak H-bonds with energies of 3-12 kcal/mol, LBHBs are proposed to exhibit energies in the 12-24 kcal/mol range [Frey, P.A., Whitt, S.A., & Tobin, J. B. (1994) Science 264, 1927-1930]. Possible roles for this LBHB both in the chemical mechanism and in the stabilization of the closed conformation of E(A-A) are discussed.
Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.
This paper proposes a strategy to translate experimental 1H NMR proton distance restraints into their corresponding heavy atom distance restraints for the purpose of protein structure prediction. The relationships between interproton distances and the corresponding heavy atom distances are determined by studying well-resolved X-ray protein structures. The data from the interproton distances of amide protons, alpha-protons, beta-protons and side chain methyl protons are plotted against the corresponding heavy atoms in scatter plots and then fitted with linear equations for lower bounds, upper bounds and optimal fits. We also transform the scatter plots into two-dimensional heat maps and three-dimensional histograms, which identify the regions where data points concentrate. The common interproton distances between amide protons, alpha-protons, beta-protons in alpha-helices, anti-parallel beta-sheets and parallel beta-sheets are also tabulated. We have found several patterns emerging from the distance relationships between heavy atom pairs and their corresponding proton pairs. All our upper bound, lower bound and optimal fit results for translating the interproton distance into their corresponding heavy atom distances are tabulated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.