Inactivation of 5-enolpyruvylshikimate 3-phosphate synthase by its substrate analogue pyruvate in the presence of sodium cyanoborohydride.
Incubation of Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase with its substrate analogue pyruvate in the presence of sodium cyanoborohydride resulted in a significant decrease in its enzyme activity. The inactivation followed pseudo-first order and saturation kinetics with a Kinact of 16.12 mM and a maximum rate of constant of 0.046 min-1. The inactivation was specifically prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus competitive inhibitor glyphosate. Upon 90% inactivation, approximately 1 mole of [14C]-label from [14C]-pyruvate was incorporated per mole of enzyme. Tryptic mapping of the modified enzyme as well as analysis of an isolated radioactive peptide indicated that Lys22 was the modified site. The results suggested that pyruvate inactivated the enzyme by forming a Schiff-base with Lys22 at the enzyme's active site.
Huynh
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Department of Protein Biochemistry, Monsanto Company, St. Louis, MO 63198.
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001894:Borohydrides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000097797:Glyphosate
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000596:Amino Acids; D001894:Borohydrides; D005998:Glycine; D008239:Lysine; D011773:Pyruvates; D019289:Pyruvic Acid; D014166:Transferases; D000097797:Glyphosate; C009282:sodium cyanoborohydride