Slowed enzymatic turnover allows characterization of intermediates by solid-state NMR.
EPSP (5-enolpyruvylshikimate-3-phosphate) synthase catalyzes condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor to the aromatic amino acids. S3P and [2-13C]POP were bound to mutant or wild type E. coli forms of the enzyme prior to lyophilization. CPMAS-echo and rotational-echo double-resonance (REDOR) NMR experiments, employing a slow catalytic EPSP synthase mutant and a long prelyophilization incubation interval, allowed our observation of the gradual formation of a strong 31P-13C coupling consistent with the well characterized tetrahedral intermediate. However, after shorter low temperature incubation intervals of substrates with mutant or wild-type enzymes, carbon CPMAS-echo NMR spectra showed the 13C label at 155 ppm, consistent with sp2 geometry of this carbon. REDOR revealed that the phosphorus of PEP was cleaved. However, phosphorus at a distance of 7.5 A was observed, due to the phosphate of a nearby bound S3P. Heating the sample allowed the reaction to progress, as shown by the diminution of the 155 ppm peak and growth of a peak at 108 ppm. The sp3 geometry implied by the 108 ppm peak strongly suggested formation of a S3P-PEP condensation product. REDOR indicated that phosphorus was still distant, but now only 6.1 (wild type) or 5.9 A (mutant) distant. We think that the early intermediates with peaks at 155 and 108 ppm are covalently bound to the enzyme. We also think that the tetrahedral intermediate that we observed was formed after product was generated.
Studelska
D R
DR
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
McDowell
L M
LM
Espe
M P
MP
Klug
C A
CA
Schaefer
J
J
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology*; D005612:Freeze Drying; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010759:Phosphorus Isotopes; D011485:Protein Binding; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013696:Temperature
D002247:Carbon Isotopes; D010759:Phosphorus Isotopes; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D010728:Phosphoenolpyruvate; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase