ALLOSTERIC REGULATION OF TRYPTOPHAN SYNTHASE - EFFECTS OF PH, TEMPERATURE, AND ALPHA-SUBUNIT LIGANDS ON THE EQUILIBRIUM DISTRIBUTION OF PYRIDOXAL 5'-PHOSPHATE-L-SERINE INTERMEDIATES

The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha(2) beta(2)-com plex from Salmonella typhimurium has been investigated by absorption a nd fluorescence spectroscopy as a function of pH, temperature, and alp ha-subunit ligands. The novel result of this study is that the equilib rium between the two major catalytic species, the external aldimine an d the alpha-aminoacrylate, is modulated by the ionization of two group s 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 i s an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associate d with the ionization of each group. At 20 degrees C, each phase accou nts for nearly half of the titration. Since the isolated beta(2)-dimer does not exhibit a pH-dependent distribution of intermediates, the al pha-beta-subunit interactions seem critical to the onset of this funct ional property of the beta-subunit. The modulation of intersubunit int eractions by the a-subunit ligands DL-alpha-glycerol 3-phosphate and p hosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alp ha-subunit ligands, the alpha-aminoacrylate Schiff base is the predomi nant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. Th e pH-dependent interconversion of catalytic intermediates here reporte d has not been previously detected because phosphate buffers have usua lly been employed in the studies of this enzyme. Our findings are disc ussed 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.