Plasticity of the tryptophan synthase active site probed by 31P NMR spectroscopy.

The functional properties of tryptophan synthase alpha2beta2 complex are modulated by a variety of allosteric effectors, including pH, monovalent cations, and alpha-subunit ligands. The dynamic properties of the beta-active site were probed by 31P NMR spectroscopy of the enzyme-bound coenzyme pyridoxal 5'-phosphate. The 31P NMR signal of the cofactor phosphate of the internal aldimine exhibits a single peak at 3.73 ppm with a line width of 12 Hz. In the presence of saturating concentrations of sodium ions, the 31P signal shifts to 3.97 ppm concomitant with a change in line width to 35 Hz. The latter indicates that sodium ions decrease the conformational flexibility of the coenzyme. In the absence of ions, lowering pH leads to the appearance of a second peak at 4.11 ppm, the intensity of which decreases in the presence of cesium ions. Addition of L-serine in the presence of sodium ions leads to the formation of the external aldimine, the first metastable catalytic intermediate. The 31P signal does not change its position, but a change in line width from 35 to 5 Hz is observed, revealing that this species is characterized by a considerable degree of rotational freedom around the coenzyme C-O bond. In the presence of L-serine and either cesium ions or the allosteric effector indole-3-acetylglycine, the accumulation of the second catalytic intermediate, alpha-aminoacrylate, is observed. The 31P signal is centered at 3.73 ppm with a line width of 5 Hz, indicating that the phosphate group of the coenzyme in the external aldimine and the alpha-aminoacrylate exhibits the same flexibility but a slightly different state of ionization. Because the alpha-aminoacrylate intermediate but not the external aldimine triggers the allosteric signal to the alpha-subunit, other portions of the beta-active site modify their dynamic properties in response to the progress of the catalytic process. A narrow line width was also observed for the quinonoid species formed by nucleophilic attack of indoline to the alpha-aminoacrylate. The 31P signal moves downfield to 4.2 ppm, indicating a possible change of the ionization state of the phosphate group. Thus, the modification of either the ionization state of the coenzyme phosphate or its flexibility or both are, at least in part, responsible for the conformational events that accompany the catalytic process.