The Tryptophan Synthase from Escherichia coli

An improved method is described for the purification of the α‐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 The standard manganese chloride and acid‐precipitation steps have been replaced by rapid and efficient chromatographic procedures.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, indolepropanol phosphate and indolebutanol phosphate have been synthesized. They are not cleaved by tryptophan synthase and are strictly competitivee 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 moieties to their respective subsites. The difference spectra generated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the α‐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 α 2 β 2 ‐complex perturbs the spectrum of pyridoxal 5′‐phosphate located in the β 2 ‐subunit. This demonstrates direct or indirect interactions between the component active sites. Binding studies by spectrophotometric titration and equilibrium dialysis with indolepropanol [ 32 P]phosphate show that there is only one binding site per equivalent of α‐subunit. Complex formation with the β 2 ‐subunit increases the affinity of the α‐subunit for indolepropanol phosphate. It is a general consequence of protein‐protein interaction in this system.