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Allosteric regulation of tryptophan synthase: effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates.

  • Peracchi, A
  • Bettati, S
  • Mozzarelli, A
  • Rossi, G L
  • Miles, E W
  • Dunn, M F
Published Article
Publication Date
Feb 13, 1996
PMID: 8639669


The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands. The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2. Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine. Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group. At 20 degrees C, each phase accounts for nearly half of the titration. Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit. The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme. Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha-aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands.


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