Intersubunit communication in tryptophan synthase by carbon-13 and fluorine-19 REDOR NMR.
The beta subunits of the 143-kDa alpha2beta2 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 beta subunit, only three pairs have labels with 13C-19F separations of less than 6 angstrom. The beta 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 beta-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 beta 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 beta subunit is changed by less than 1 angstrom.
McDowell
L M
LM
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Lee
M
M
McKay
R A
RA
Anderson
K S
KS
Schaefer
J
J
D002247:Carbon Isotopes; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005461:Fluorine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000378:metabolism*; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002247:Carbon Isotopes; D011994:Recombinant Proteins; D005461:Fluorine; D012694:Serine; D014367:Tryptophan Synthase