Kinetics of cooperative ligand binding to the apo.beta.2 subunit of tryptophan synthase and its modulation by the .alpha. subunit

Abstract

The different binding mechanisms of pyridoxine 5''-phosphate and N-phosphopyridoxyl-L-serine were investigated by kinetic studies with rapid reaction techniques. Pyridoxine 5''-phosphate binds in a single rapid step to the .alpha.2apo.beta.2 complex of [Escherichia coli] tryptophan synthase and in a single slow step to the nicked apo.beta.2 subunit that is obtained by limited proteolysis with trypsin. Pyridoxine 5''-phosphate and N-phosphopyridoxyl-L-serine bind to the apo.beta.2 subunit with a comparatively slow binding step, followed by an even slower isomerization reaction. These findings are consistent with the nonexclusive concerted mechanism of cooperative binding but cannot be explained by the simple sequential mechanism. A quantitative fit of the rate and equilibrium data to the concerted mechanism generally yielded the pertinent rate and equilibrium constants. The same value of L0 = [T0]/[R0] = 200 .+-. 50 simultaneously satisfies the data obtained with 3 different ligands. The comparison of the mechanisms of ligand binding to the 3 states of the apo.beta.2 subunit suggests that the .alpha.2apo.beta.2 complex to the high-affinity R state and the nicked apo.beta.2 subunit is similar to the low-affinity T state of the apo.beta.2 subunit. The slow isomerization involved in the cooperative binding of the ligands to the intact apo.beta.2 subunit is discussed in terms of local and concerted conformational changes involving the 2 autonomously folding domains of the .beta. protomer.