Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis.
Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.
Padgette
S R
SR
Plant Molecular Biology Group, Monsanto Company, Chesterfield, Missouri 63198.
Huynh
Q K
QK
Aykent
S
S
Sammons
R D
RD
Sikorski
J A
JA
Kishore
G M
GM
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*; D000097797:Glyphosate
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D005998:Glycine; D012765:Shikimic Acid; D014166:Transferases; D000097797:Glyphosate; C042786:shikimic acid-3-phosphate