title: Arginine chemical modification of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase.
abstract: Reaction of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) with the arginine reagents phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPGO) leads to inactivation of the enzyme. Inactivation with HPGO leads to modification of approximately 3 mol of arginine per mole of enzyme. The modification reaction follows pseudo-first-order kinetics with a t1/2 of 1 min at 5 mM p-hydroxyphenylglyoxal in 0.1 M triethanolamine HCl, pH 7.8. By titration of HPGO-modified enzyme with 5,5'-bis(dithio-2-nitrobenzoic acid), the possibility of cysteine modification by the arginine reagent was ruled out. While shikimate 3-phosphate (S3P) afforded partial protection to the enzyme against inactivation by HPGO, complete protection could be obtained by using a mixture of S3P and glyphosate. Under the latter conditions, only 1 mol arginine was modified per mole of enzyme. This pattern of reactivity suggests that two arginines may be involved in the binding of S3P and glyphosate to EPSP synthase. A third reactive arginine appears to be nonessential for EPSPS activity. Labeling of EPSP synthase with [14C]phenylglyoxal, peptic digestion, HPLC mapping, and amino acid sequencing indicate that Arg-28 and Arg-131 are two of the reactive arginines labeled with [14C]PGO.
authors.affiliation: Plant Molecular Biology and Chemistry Groups, Biological Sciences, Monsanto.
authors.forename: S R
authors.initials: SR
authors.lastname: Padgette
authors.forename: C E
authors.initials: CE
authors.lastname: Smith
authors.forename: Q K
authors.initials: QK
authors.lastname: Huynh
authors.forename: G M
authors.initials: GM
authors.lastname: Kishore
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001120:Arginine; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008969:Molecular Sequence Data; D010658:Phenylglyoxal / Q000494:pharmacology; D010944:Plants; D013438:Sulfhydryl Compounds; D014166:Transferases / Q000037:antagonists & inhibitors
chemical_list: D013438:Sulfhydryl Compounds; D001120:Arginine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D010658:Phenylglyoxal