The tryptophan synthase from Escherichia coli. An improved purification procedure for the alpha-subunit and binding studies with substrate analogues.
An improved method is described for the purification of the alpha-subunit of tryptophan synthase from Escherichia coli. The standard manganese chloride and acid-precipitation steps have been replaced by rapid and efficient chromatographic procedures. Indoleethanol phosphate, indoleprapanol phosphate and indolebutanol phosphate have been synthesized. They are not cleaved by tryptophan synthase and are strictly competitive inhibitors versus indoleglycerol phosphate. The inhibition constant decreases as the number of methylene groups in the side chain increases. This may reflect an improved accommodation of the indole and phosphate moienerated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the alpha-subunit are very similar. This reflects the transfer of the indole moiety to an hydrophobic environment within the active center. The binding of indolepropanol phosphate to the alpha2beta2-complex perturbs the spectrum of pyridoxal 5'-phosphate located in the beta2-subunit. This demonstrates direct or indirect interactions between the component active sites. Bind studies by spectrophotometric titration and equilibrium dialysis with indolepropanol [32P]phosphate show that there is only one binding site per equivalent of alpha-subunit. Complex formation with the beta2-subunit increases the affinity of the alpha-subunit for indolepropanol phosphate, It is a general consequence of protein-protein interaction in this system.
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007211:Indoles; D008024:Ligands; D046911:Macromolecular Substances; D008433:Mathematics; D009943:Organophosphorus Compounds; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014367:Tryptophan Synthase / Q000302:isolation & purification* / Q000378:metabolism
D007211:Indoles; D008024:Ligands; D046911:Macromolecular Substances; D009943:Organophosphorus Compounds; D014367:Tryptophan Synthase