5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411.
In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.
Huynh
Q K
QK
Department of Protein Biochemistry, Monsanto Company, St. Louis, MO 63198.
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D011773:Pyruvates / Q000494:pharmacology*; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D014166:Transferases / Q000037:antagonists & inhibitors*; D014357:Trypsin / Q000494:pharmacology
D008024:Ligands; D011773:Pyruvates; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; C017092:bromopyruvate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D014357:Trypsin; D008239:Lysine; D003545:Cysteine