On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase.
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.
Jakeman
D L
DL
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
Mitchell
D J
DJ
Shuttleworth
W A
WA
Evans
J N
JN
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D002384:Catalysis; D004789:Enzyme Activation; D007711:Klebsiella pneumoniae; D019906:Nuclear Magnetic Resonance, Biomolecular; D012996:Solutions; D013816:Thermodynamics
D012996:Solutions; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase