PMID:1348247
| Citation |
Stephens, CM and Bauerle, R (1992) Essential cysteines in 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Analysis by chemical modification and site-directed mutagenesis of the phenylalanine-sensitive isozyme. J. Biol. Chem. 267:5762-7 |
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| Abstract |
The phenylalanine-sensitive isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli was inactivated by the sulfhydryl modifying reagents 5,5-dithiobis-(2-nitrobenzoate), bromopyruvate, and N-ethylmaleimide and protected from inactivation by the presence of its metal activator, Mn2+, and substrate, phosphoenolpyruvate. Inactivation by 5,5-dithiobis-(2-nitrobenzoate) was correlated with modification of two of the seven cysteine sulfhydryls of the enzyme monomer. The kinetics of 5,5-dithiobis-(2-nitrobenzoate) modification were altered significantly and distinctively by both substrates (phosphoenolpyruvate and erythrose 4-phosphate), by Mn2+, and by L-phenylalanine, suggesting that ligand binding has significant effects on the conformation of the enzyme. Site-directed mutagenesis was used to create multiple substitutions at the two invariant cysteine residues of the polypeptide, Cys-61 and Cys-328. Analysis of purified mutant enzymes indicated that Cys-61 is essential for catalytic activity and for metal binding. Cys-328 was found to be nonessential for catalytic activity, although mutations at this position had significant negative effects on Vmax, KmMn, and KmPEP. |
| Links | |
| Keywords |
3-Deoxy-7-Phosphoheptulonate Synthase/genetics; 3-Deoxy-7-Phosphoheptulonate Synthase/metabolism; Amino Acid Sequence; Base Sequence; Cysteine; Dithionitrobenzoic Acid/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Isoenzymes/genetics; Isoenzymes/metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Phenylalanine/pharmacology; Plasmids; Pyruvates/pharmacology; Sulfhydryl Reagents/pharmacology |
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