Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by carbon-13 NMR spectroscopy

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Abstract
The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]- and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., and Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., and Johnson, K. A. (1988) J. Am. Chem. Soc 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [313C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group of 4-hydroxy-4-methylketoglutarate. The resonance at 25.5 ppm, which was found to correlate with a 1H resonance at 1.16 ppm, could be substantially reduced by preincubating the enzyme with the inhibitor glyphosate and could be isolated free from the protein by ultrafiltration. This has been interpreted in terms of the same enzyme-free tetrahedral intermediate that gave rise to the resonance at 110.9 ppm with [2-13C]PEP and porposed by Levin and Sprison. No 1H correlation has been detected for the 13C resonance at 24.5 ppm due to its large line width (100 Hz), but it has been interpreted as arising from the same enzyme-bound intermediate which gave rise to the resonance at 107.2 ppm with [2-13C]PEP.