title: Site-directed mutagenesis of Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase: Lys-23 is essential for substrate binding.
abstract: Chemical modification of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine), with pyridoxal 5'-phosphate suggested that Lys-22 (equivalent to Lys-23 of the Petunia hybrida enzyme) is a potential active site residue (Huynh, Q. K., Kishore, G. M., and Bild, G. S. (1988) J. Biol. Chem. 263, 735-739). To investigate the possible role of this residue in the reaction mechanism, we have used site-directed mutagenesis to replace Lys-23 of the P. hybrida enzyme with 3 other amino acid residues: Ala, Glu, and Arg. Analysis of these mutant enzymes indicates that of these only the Lys-23 to Arg mutant enzyme is active; the other two replacements (Ala and Glu) result in inactivation of the enzyme. Two of the mutant enzymes (Lys-23 to Arg and Ala) were purified to homogeneity and characterized. The purified Lys-23 to Arg mutant enzyme is less sensitive than the wild type enzyme to pyridoxal 5'-phosphate. It showed identical Km values for substrates and a 5-fold higher I50 value for glyphosate in comparison with those from the wild type enzyme. Binding studies using fluorescence measurements revealed that the substrate shikimate 3-phosphate and glyphosate were able to bind the purified Lys-23 to Arg mutant enzyme but not to the purified catalytically inactive Lys-23 to Ala mutant enzyme. The above results suggest that the cationic group at position 23 of the enzyme may play an important role in substrate binding.
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D000409:Alanine; D019883:Alkyl and Aryl Transferases; D001120:Arginine; D001665:Binding Sites; D001667:Binding, Competitive; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006540:Herbicides; D008239:Lysine; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014166:Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D000409:Alanine; D019883:Alkyl and Aryl Transferases; D001120:Arginine; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D006540:Herbicides; D008239:Lysine; D010728:Phosphoenolpyruvate; D011732:Pyridoxal Phosphate; D012765:Shikimic Acid; D014166:Transferases; D000097797:Glyphosate; C042786:shikimic acid-3-phosphate