Phosphorus-31 nuclear magnetic resonance study of pyridoxal 5'-phosphate binding to Escherichia coli tryptophan synthase.

The pyridoxal phosphate dependent bienzyme complex tryptophan synthase from Escherichia coli has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In both the isolated beta 2 subunit and the fully assembled alpha 2 holo beta 2 complex, the pyridoxal phosphate 31P chemical shift is pH independent, indicating that the phosphate group of the cofactor is excluded from interaction with water and fixed in its dianionic form to the protein moiety. The line width of the phosphate signal of the coenzyme in the beta 2 subunit is unusually large pointing to different equilibrium conformations of the protein. In the native holo complex, however, the corresponding line width may be interpreted to result from moderate mobility of the cofactor leading to a higher rate of the transaldimination step of the catalytic mechanism.