title: Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli.
abstract: 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.
mesh_terms: D001059:Apoproteins; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001059:Apoproteins; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase