AN EPSP SYNTHASE INHIBITOR JOINING SHIKIMATE 3-PHOSPHATE WITH GLYPHOSATE - SYNTHESIS AND LIGAND-BINDING STUDIES

A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its he rbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calo rimetric and P-31 NMR spectroscopic studies of enzyme-bound 4, now pro vides a more precise quantitative definition for the molecular interac tions of 4 with this enzyme. The very poor binding, relative to 4, dis played by the 3-dephospho analog 12 is indicative that 4 has a specifi c interaction with the S3P site. A comparison of K-i(calc) for 12 vers us the K-i(app) for 4 indicates that the 3-phosphate group in 4 contri butes about 4.8 kcal/mol to binding. This compares well with the 5.2 k cal/mol which the 3-phosphate group in S3P contributes to binding. Iso thermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed K-d Of 0.53 +/- 0.04 mu M. As such, 4 binds only 3-f old weaker than glyphosate and about 150-fold better than N-methylglyp hosate. Consequently, 4 represents the most potent N-alkylglyphosate d erivative identified to date. However, the resulting thermodynamic bin ding parameters clearly demonstrate that the formation of EPSPS . 4 is entropy driven like S3P. The binding characteristics of 4 are fully c onsistent with a primary interaction localized at the S3P subsite. Fur thermore, P-31 NMR studies of enzyme-bound 4 confirm the expected inte raction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS 4 is in the opposite dir ection than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is in complete overlap at the glyphosate phosphonate subsite. As a glyphosat e analog inhibitor, the potency of 4 most likely arises from predomina nt interactions which occur outside the normal glyphosate binding site . Consequently, 4 is best described as an S3P-based substrate-analog i nhibitor. These combined results corroborate the previous kinetic mode l [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, interactio n at the expected glyphosate phosphonate or phosphoenolpyruvate-P-i su bsites.