Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements.
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)
Anderson
K S
KS
Technology Division, Monsanto Agricultural Company, St. Louis, Missouri 63167.
Sikorski
J A
JA
Johnson
K A
KA
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008962:Models, Theoretical; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014166:Transferases / Q000378:metabolism*
D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase