Abstract
The pyridoxal 5'-phosphate-dependent beta-subunit of the tryptophan synthase alpha(2)beta(2) complex catalyzes the condensation of L-serine with indole to form L-tryptophan. The first stage of the reaction is a beta-elimination that involves a very fast interconversion of the internal aldimine in a highly fluorescent L-serine external aldimine that decays, via the alpha-carbon proton removal and beta-hydroxyl group release, to the alpha-aminoacrylate Schiff base. This reaction is influenced by protons, monovalent cations, and alpha-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 k(1)) 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 k(1) exhibits a bell shaped profile characterized by a pK(a1) of about 6 and a pK(a2) of about 9, whereas in the presence of cesium ions, the pH dependence exhibits a saturation profile characterized by a single pK(a) of 9. The presence of the allosteric effector indole acetylglycine increases the rate of reaction without altering the pH profile and pK(a) values. By combining structural information for the internal aldimine, the external aldimine, and the alpha-aminoacrylate with kinetic data on the wild type enzyme and beta-active site mutants, we have tentatively assigned pK(a1) to betaAsp-305 and pK(a2) to betaLys-87. The loss of pK(a1) 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 beta-subunit.
Highlights
The pyridoxal 5-phosphate-dependent -subunit of the tryptophan synthase ␣22 complex catalyzes the condensation of L-serine with indole to form L-tryptophan
The same reaction was monitored by single wavelength stopped flow by recording the fluorescence emission of the external aldimine, upon excitation at 420 nm, in the absence (Fig. 2a) and presence of 250 mM NaCl (Fig. 2b), 100 mM KCl (Fig. 2c), and 100 mM CsCl (Fig. 2d) at pH values between 6 and 8.8
In the first stage of the reaction catalyzed by tryptophan synthase (TS), there are at least three steps that may require the presence of a proton donor/acceptor residue (Scheme 1)
Summary
The pyridoxal 5-phosphate-dependent -subunit of the tryptophan synthase ␣22 complex catalyzes the condensation of L-serine with indole to form L-tryptophan. The first stage of the reaction is a -elimination that involves a very fast interconversion of the internal aldimine in a highly fluorescent L-serine external aldimine that decays, via the ␣-carbon proton removal and -hydroxyl group release, to the ␣-aminoacrylate Schiff base. This reaction is influenced by protons, monovalent cations, and ␣-subunit ligands that modulate the distribution between open and closed conformations. The release of L-tryptophan completes the catalytic cycle regenerating the internal aldimine
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