Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.
Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006540:Herbicides; D007700:Kinetics; D008969:Molecular Sequence Data; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D014166:Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000097797:Glyphosate
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D005022:Ethyldimethylaminopropyl Carbodiimide; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D006540:Herbicides; D012765:Shikimic Acid; D014166:Transferases; D000097797:Glyphosate; C022010:glycine ethyl ester; C042786:shikimic acid-3-phosphate