Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogs

Abstract

An improved purification procedure for the .alpha.2.beta.2 complex of tryptophan synthase from E. coli was developed. It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously. Inhibition studies, equilibrium dialysis and spectrophotometric titration were used to study the binding of both pyridoxal phosphate analogs and bisubstrate analogs. Pyridoxine 5''-phosphate and N-phospho-pyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5''-phosphate and N-phosphopyridoxyl-L-alanine do not. N-phosphopyridoxyl-L-tryptophan is bound weakly, although L-tryptophan binds strongly to the .alpha.2holo.beta.2 complex. It is likely that either differences in protonation or in geometry are responsible for the low affinity of the bisubstrate analogs in comparison to that of the external aldimines of either L-serine or L-tryptophan with pyridoxal 5''-phosphate. As previously found with pyridoxal 5''-phosphate, pyridoxine 5''-phosphate and N-phosphopyridoxyl-L-serine bind noncooperatively to 2 identical binding sites in the .alpha.2apo.beta.2 complex. The same ligands bind with positive cooperativity to 2 binding sites in the apo.beta.2 subunit. Because the analogs mimic the binding behavior of pyridoxal 5''-phosphate to both proteins, the internal aldimine of pyridoxal 5''-phosphate to the lysine amino group contributes only to the strength of that binding. The nicked apo.beta.2 subunit, which is produced by limited proteolysis with trypsin, binds pyridoxine 5''-phosphate noncooperatively to 2 identical sites. The loop of polypeptide chain connecting the 2 autonomous domains of folding must be intact for enzyme activity, binding of the .alpha. subunit and cooperative binding of pyridoxine 5''-phosphate.