title: Photo-oxidation of 5-enolpyruvoylshikimate-3-phosphate synthase from Escherichia coli: evidence for a reactive imidazole group (His385) at the herbicide glyphosate-binding site.
abstract: Photo-oxidation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the non-selective herbicide glyphosate (N-phosphonomethylglycine), in the presence of pyridoxal 5'-phosphate resulted in irreversible inactivation of the enzyme. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact. of 50 microM. The inactivation is specifically prevented by preincubation of the enzyme with the combination of shikimate 3-phosphate and glyphosate. Increasing glyphosate concentration during preincubation resulted in a decreasing rate of inactivation. On 95% inactivation, approximately one histidine per molecule of enzyme was oxidized. Tryptic mapping of the enzyme modified in the absence and presence of shikimate 3-phosphate and glyphosate as well as analyses of the histidine content in the isolated peptides indicated that His385, in the peptide Asn383-Asp-His-Arg386, was the site of oxidation. These results suggest that His385 is the most accessible reactive imidazole group under these conditions and is located close to the glyphosate-binding site.
authors.affiliation: Department of Protein Biochemistry, Monsanto Company, St. Louis, MO 63198.
authors.forename: Q K
authors.initials: QK
authors.lastname: Huynh
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006540:Herbicides / Q000378:metabolism*; D006639:Histidine / Q000032:analysis*; D007093:Imidazoles / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010777:Photochemistry; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D005998:Glycine; D006540:Herbicides; D006639:Histidine; D007093:Imidazoles; D014166:Transferases; D000097797:Glyphosate