Abstract
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.
Highlights
From the PlantMolecular Biology and §Chemistry Groups, Biological Sciences, Corporate Research and Development StafL Monsanto Company, and the TTechnology Division, Monsanto Agricultural Company, Chesterfield, Missouri 63198
In view of the lack of information on the gest that the cysteine thiol is proximal to the binding reactivity of cysteine residues of EPSP synthase, the studies site for thesleigands
Tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines havebeen identified as Cys408and C Y S ~T~he~c.ysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to beCys408
Summary
5-Enolpyruvylshikimate-3-phosphateSynthase and Their Nonessentiality for Enzymatic Catalysis*. Reaction of 5-enolpyruvylshikimate-3-phosphate vylshikimate3-phosphate from shikimate3-phosphateand synthase of Escherichia coli with the thiol reagent phosphoenolpyruvate (P-enolpyruvate)(2). The sensitivity of EPSP synthe active site-reactive cysteineof E. coli 5-enolpyru- thases to reagents such as bromopyruvate and N-ethylmalvylshikimate-3-phosphate synthase for either catalysiesimide (NEM) hasraised the question of whether an essential or substrate binding. In view of the lack of information on the gest that the cysteine thiol is proximal to the binding reactivity of cysteine residues of EPSP synthase, the studies site for thesleigands. Tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines havebeen identified as Cys408and C Y S ~T~he~c.ysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to beCys408. Thase,’ EC 2.5.1.19), an enzyme of the shikimate pathway in plants and bacteria, catalyzes the formation of 5-enolpyru-
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