Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli.

An improved purification procedure for the beta2 subunit of tryptophan synthase from from Escherichia coli has led to an essentially pure and stable preparation with a specific enzymatic activity that is 30% higher than the previously reported maximum value. Sedimentation analysis shows that the apo-beta2 subunit is monodisperse and dimeric down to a concentration of 0.02 mg of protein/ml. The binding of pyridoxal 5'-phosphate (pyridoxal-P) to the apo-beta2 subunit and to the alpha2-apo-beta2 complex was studied by equilibrium dialysis and spectroscopic titration. Both the beta2 subunit and the alpha2beta2 complex bind 2 mol of pyridoxal-P with no unspecific binding observable at higher concentrations of pyridoxal-P. The binding of pyridoxal-P to the apo-beta2 subunit is cooperative (Hill coefficient nH = 1.7). The data have been fitted to the Adair equation, yielding the apparent microscopic dissociation constants for the complexes with one and two bound ligand molecules. They differ by a factor of 38, suggesting that the apo- and holo-beta2 subunits have distinct conformations. The binding of pyridoxal-P to the alpha2-apo-beta2 complex is noncooperative with a value of the dissociation constant intermediate between the two values of the beta2 subunit. This finding suggests that the alpha subunit may stabilize a third conformational state of the beta2 subunit.