The pyridoxal 5′-phosphate-dependent β-subunit of the tryptophan synthase α2β2 complex catalyzes the condensation of L-serine with indole to form L-tryptophan. The first stage of the reaction is a β-elimination that involves a very fast interconversion of the internal aldimine in a highly fluorescent L-serine external aldimine that decays, via the α-carbon proton removal and β-hydroxyl group release, to the α-aminoacrylate Schiff base. This reaction is influenced by protons, monovalent cations, and α-subunit ligands that modulate the distribution between open and closed conformations. In order to identify the ionizable residues that might assist catalysis, we have investigated the pH dependence of the rate of the external aldimine decay by rapid scanning UV-visible absorption and single wavelength fluorescence stopped flow. In the pH range 6-9, the reaction was found to be biphasic with the first phase (rate constants k1) accounting for more than 70% of the signal change. In the absence of monovalent cations or in the presence of sodium and potassium ions, the pH dependence of k1 exhibits a bell shaped profile characterized by a pKa1 of about 6 and a pKa2 of about 9, whereas in the presence of cesium ions, the pH dependence exhibits a saturation profile characterized by a single pKa of 9. The presence of the allosteric effector indole acetylglycine increases the rate of reaction without altering the pH profile and pKa values. By combining structural information for the internal aldimine, the external aldimine, and the α-aminoacrylate with kinetic data on the wild type enzyme and β-active site mutants, we have tentatively assigned pKa1 to βAsp-305 and pKa2 to βLys-87. The loss of pKa1 in the presence of cesium ions might be due to a shift to lower values, caused by the selective stabilization of a closed form of the β-subunit.
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