Slowed enzymatic turnover allows characterization of intermediates by solid-state NMR

Biochemistry
Daniel R StudelskaJ Schaefer

Abstract

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 ph...Continue Reading

References

Jan 1, 1994·Annual Review of Physical Chemistry·S J Opella
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Dec 1, 1996·Solid State Nuclear Magnetic Resonance·C A KlugJ Schaefer
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Citations

Mar 21, 1998·Current Opinion in Biotechnology·K A Johnson
Dec 7, 2007·Nature Structural & Molecular Biology·Sandra ChimonYoshitaka Ishii
Mar 12, 2004·Annual Review of Plant Physiology and Plant Molecular Biology·Klaus M. Herrmann, Lisa M. Weaver
Dec 8, 2004·Archives of Biochemistry and Biophysics·Karen S Anderson
Jun 30, 2000·Journal of Magnetic Resonance·B ReifR G Griffin
Apr 8, 2019·The Journal of Chemical Physics·Mukul G JainVipin Agarwal
Sep 30, 2005·Journal of the American Chemical Society·Sandra Chimon, Yoshitaka Ishii

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