Intersubunit Communication in Tryptophan Synthase by Carbon-13 and Fluorine-19 REDOR NMR

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Abstract
The β subunits of the 143-kDa α2β2 tetrameric enzyme tryptophan synthase have been labeled by l-[ring-4-19F]phenylalanine and l-[phenol-4-13C]tyrosine in an effort to monitor the positions of these residues on ligand binding. Of the 13 phenylalanine and 11 tyrosine residues in the β subunit, only three pairs have labels with 13C−19F separations of less than 6 Å. The β-subunit residues Tyr279 and Phe280 (each members of one of the three Tyr−Phe proximate pairs) have been suggested as possible conformational gates on ligand binding. The 188-MHz 19F NMR spectrum of the microcrystalline, double-labeled enzyme complex has five resolved lines under 5-kHz magic-angle spinning and 80-kHz proton dipolar decoupling. The distribution of β-subunit 19F isotropic shifts is altered by addition of l-[3-13C]serine to the mother liquor in contact with the microcrystals, consistent with a conformational rearrangement. The 13C label from serine is detected at 28 ppm as a methyl tautomer of bound aminoacrylate. The change in aromatic 19F chemical shifts on binding of serine indicates an alteration in local electric field gradients within the β subunit. However, rotational-echo double-resonance 13C NMR (with 19F dephasing) shows that the average 13C−19F distance for the three phenylalanine−tyrosine proximate pairs in the β subunit is changed by less than 1 Å.