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 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.

Steady-state kinetic evaluation of the reverse reaction for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase.

Recently it has been found that the kinetic mechanism for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) in the forward direction is random with synergistic binding of substrates and inhibitors (K. J. Gruys, M. C. Walker, and J. A. Sikorski, 1992, Biochemistry 31, 5534). This work, however, did not address the reverse reaction with 5-enolpyruvoylshikimate-3-phosphate (EPSP) and phosphate (Pi) as substrates where a similar question of random versus ordered addition of substrates remained. Previous transient-state kinetic results led to a proposal for an equilibrium-ordered mechanism, where binding of EPSP occurs first followed by Pi (K. S. Anderson, and K. A. Johnson, 1990, Chem. Rev. 90, 1131). Steady-state kinetic results of the reverse reaction presented here suggest that, like the forward reaction, addition of substrates occurs randomly. Initial velocity studies with EPSP and Pi show a normal intersecting pattern in the reciprocal plots, consistent with a random or steady-state-ordered mechanism, but not with equilibrium-ordered addition of substrates. Inhibition of the EPSPS reverse reaction by 5-amino-S3P or the S3P-glyphosate hybrid molecule gave the expected competitive patterns versus EPSP, but mixed noncompetitive patterns versus Pi. These results also disfavor an equilibrium-ordered model, but again are consistent with a random or steady-state-ordered mechanism. A more quantitative mechanistic analysis of the inhibition data to determine the true rather than apparent Ki values provides evidence for a random over a steady-state-ordered addition of substrates. These results in combination with previous findings lead to the conclusion that the mechanism is random addition of EPSP and Pi since it is the only possible model for substrate addition that is consistent with the cumulative data from all kinetic (transient- as well as steady-state) and direct binding studies.