Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy.
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 [2-13C]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., & 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., & 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 [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 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 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)
Barlow
P N
PN
Dyson Perrins Laboratory, University of Oxford, U.K.
Appleyard
R J
RJ
Wilson
J O
JO
Evans
J N
JN
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D002247:Carbon Isotopes; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014166:Transferases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D002247:Carbon Isotopes; D011773:Pyruvates; C062226:4,5-dideoxyshikimic acid 3-phosphate; D012765:Shikimic Acid; D010728:Phosphoenolpyruvate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D010744:Phosphoric Monoester Hydrolases