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-C-13]PEP 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 P-31-C-13 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 C-13 label at 155 ppm, consistent with sp(2) geometry of this carbon. REDOR revealed that the phosphorus of PEP was cleaved, However, phosphorus at a distance of 7.5 Angstrom 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 sp(3) 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 Angstrom (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.