J. Biol. Chem., Vol. 265, Issue 12, 6700-6704, Apr, 1990
Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3- phosphate synthase by o-phthalaldehyde
QK Huynh
Department of Biological Sciences, Monsanto Co., St. Louis, Missouri 63198.
In order to identify the essential reactive amino acid residues of 5-
enolpyruvoylshikimate-3-phosphate synthase, a target for the nonselective
herbicide glyphosphate (N-phosphonomethylglycine), chemical modification
studies with o-phthalaldehyde were undertaken. Incubation of the enzyme
with the reagent resulted in a time-dependent loss of enzyme activity. The
inactivation followed first-order and saturation kinetics with a Kinact of
25 microM and a maximum rate constant of 0.34 min-1. The inactivation was
prevented by preincubation of the enzyme with the substrates shikimate
3-phosphate, 5- enolpyruvoylshikimate 3-phosphate, or by a combination of
shikimate 3- phosphate plus glyphosate, but not by phosphoenolpyruvate or
glyphosate alone. Absorbance and fluorescence spectra studies indicate that
complete inactivation of the enzyme resulted from the formation of two
isoindole derivatives per molecule of enzyme. Tryptic mapping of the enzyme
modified in the absence of shikimate 3-phosphate and glyphosate resulted in
the isolation of two peptides which were not found for the enzyme modified
in the presence of shikimate 3-phosphate and glyphosate. Analyses of these
two peptides indicate that Lys-22 and Lys- 340 were the modified sites. The
amino acid sequences around these residues are conserved in bacterial,
fungal, as well as plant enzymes, suggesting that these regions may
constitute part of the enzyme active site.
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