Affinity chromatography of tryptophan synthase from Escherichia coli. Systematic studies with immobilized tryptophanol phosphate.
Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase. A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme. The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding. The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions. The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands. Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly. Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e. via the active site of alpha subunit. L-Serine is a non-competitive inhibitor of binding. These results are discussed with regard to the composite-active-site hypothesis [T. E. Creighton (1970) Eur. J. Biochem, 13, 1--10]. Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations. Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine. At the low concentrations of the complex found in crude extracts of wild-type E. coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex.
D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000031:analogs & derivatives*; D014367:Tryptophan Synthase / Q000302:isolation & purification* / Q000378:metabolism
D008024:Ligands; D046911:Macromolecular Substances; C020536:tryptophanol phosphate; D014364:Tryptophan; D014367:Tryptophan Synthase