Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit.
The different binding mechanisms of pyridoxine 5'-phosphate and N-phophopridoxyl-L-serine have been investigated by kinetic studies with rapid reaction techniques. Pyridoxine 5'-phosphate binds in a single rapid step to the alpha 2 apo beta 2 complex and in a single slow step to the nicked apo beta 2 subunit that is obtained by limited proteolysis with trypsin. Both pyridoxine 5'-phosphate and N-phosphopyridoxyl-L-serine bind to the apo beta 2 subunit with a comparatively slow binding step, followed by an event slower isomerization reaction. These findings are consistent with 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. In particular, the same value of L0 = [T0]/[R0] = 200 +/- 50 simultaneously satisfies the data obtained with three different ligands. The comparison of the mechanisms of ligand binding to the three states of the apo beta 2 subunit suggests that the alpha 2 apo beta 2 complex is similar 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 isonerization 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 two autonomously folding domains of the beta protomer.
D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011736:Pyridoxine / Q000031:analogs & derivatives / Q000494:pharmacology; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D014357:Trypsin; D014367:Tryptophan Synthase / Q000378:metabolism*
D001051:Apoenzymes; D046911:Macromolecular Substances; D012694:Serine; D011732:Pyridoxal Phosphate; C026853:N-phosphopyridoxyl-L-serine; D014357:Trypsin; D014367:Tryptophan Synthase; D011736:Pyridoxine; C024593:pyridoxine 5-phosphate