Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements

Abstract

The binding of substrates and the herbicide N-(phosphonomethyl)glycine (glyphosate) to enolpyruvoylshikimate-3-phosphate (EPSP) synthase was evaluated by stopped-flow and equilibrium fluorescence measurements. Changes in protein fluorescence were observed upon the binding of EPSP and upon the formation of the enzyme-shikimate 3-phosphate-glyphosate ternary complex; no change was seen with either shikimate 3-phosphate (S3P) or glyphosate alone. By fluorescence titrations, the dissociation constants were determined for the formation of the enzyme binary complexes with S3P (Kd,S = 7 +/- 1.2 microM) and EPSP (Kd,EPSP = 1 +/- 0.01 microM). The dissociation constant for S3P was determined by competition with EPSP or by measurements in the presence of a low glyphosate concentration. At saturating concentrations of S3P, glyphosate bound to the enzyme--S3P binary complex with a dissociation constant of 0.16 +/- 0.02 microM. Glyphosate did not bind significantly to free enzyme, so the binding is ordered with S3P binding first: (formula; see text) where S refers to S3P, G refers to glyphosate, and E.S.G. represents the complex with altered fluorescence. The kinetics of binding were measured by stopped-flow fluorescence methods. The rate of glyphosate binding to the enzyme--S3P complex was k2 = (7.8 +/- 0.2) X 10(5) M-1 s-1, from which we calculated the dissociation rate k-2 = 0.12 +/- 0.02 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

“…titrations (21). The value Kd,phosphate= 4.8 mM, was similar to values measured previously with wild-type AroA, which range from 0.…”

“…The reaction mechanisms of both MurA and AroA have been studied intensively with pre-steady state experiments (11,14,15), site directed mutagenesis (11), substrate analogues (16)(17)(18), x-ray crystallography (7,19,20), fluorescence titration (21) and NMR experiments (6,22). Their mechanisms are similar in several aspects.…”

“…This oxacarbenium ion exists long enough for the C3 to rotate, and one of the methyl protons to be removed during deprotonation, allowing tritium to remain in PEP. The formation of the oxacarbenium ion also requires a conformational change in AroA, which was monitored by fluorescence titrations (21). showing that tritium can be incorporated into C3 of PEP.…”

Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

“…The cuvette held a 3-ml sample containing 0.15 M DgkB, 1/10,000 dilution of SYPRO Orange DMSO stock (Invitrogen) in 50 mM HEPES, pH 7.4, 150 mM LiCl. Intrinsic tryptophan fluorescence intensity (27) was the second method of detecting conformational changes in DgkB. Because DgkB does not contain tryptophan, the DgkB[Y163W] mutant was constructed to place a tryptophan reporter residue on the ␤8 -␣6 loop adjacent to Lys 165 .…”

Closing down on glyphosate inhibition—with a new structure for drug discovery

“…Using limited tryptic digestion and matrix-assisted laser desorption ionizationtime-of-flight mass spectroscopy, Krekel et al (14) have suggested that binding of S3P in combination with glyphosate also stabilizes EPSPS in a closed conformation. In addition, fluorescence spectroscopy studies imply that the association of glyphosate with the EPSPS-S3P binary complex induces a substantial conformational change in the enzyme (15). In a separate study using radiolabeled plant PreEPSPS (mature enzyme with a short chloroplast transit peptide), Della-Cioppa and Kishore (16) have shown that the native precursor protein is transported into chloroplasts, whereas a PreEPSPS-S3P-glyphosate ternary complex was not readily transported into the chloroplast stroma, suggesting that the ternary complex in its closed form is probably hindered in temporarily undergoing a conformational change to facilitate the process of importation across the chloroplast membrane.…”