title: An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.
abstract: A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described as an S3P-based substrate-analog inhibitor. These combined results corroborate the previous kinetic model [Gruys, K. J., Marzabadi, M. R., Pansegrau, P. D., & Sikorski, J. A. (1993) Arch. Biochem. Biophys. 304, 345-351], which suggested that 4 interacts well with the S3P subsite but has little, if any, interaction at the expected glyphosate phosphonate or phosphoenolpyruvate-Pi subsites.
authors.affiliation: Ceregen and Monsanto Corporate Research, Units of Monsanto Company, St. Louis, Missouri 63198.
authors.forename: M R
authors.initials: MR
authors.lastname: Marzabadi
authors.forename: K J
authors.initials: KJ
authors.lastname: Gruys
authors.forename: P D
authors.initials: PD
authors.lastname: Pansegrau
authors.forename: M C
authors.initials: MC
authors.lastname: Walker
authors.forename: H K
authors.initials: HK
authors.lastname: Yuen
authors.forename: J A
authors.initials: JA
authors.lastname: Sikorski
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D001665:Binding Sites; D002151:Calorimetry; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D005998:Glycine / Q000031:analogs & derivatives*; D006540:Herbicides; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D014166:Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D004791:Enzyme Inhibitors; D005998:Glycine; D006540:Herbicides; D007202:Indicators and Reagents; D008024:Ligands; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D012765:Shikimic Acid; D014166:Transferases; D000097797:Glyphosate; C082466:N-(carboxymethyl)-N-(phosphonomethyl)-5-aminoshikimate-3-phosphate; C042786:shikimic acid-3-phosphate