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.
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.
Department of Biology, University of Virginia, Charlottesville 22901.
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Stephens
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Bauerle
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine / Q000494:pharmacology*; D010957:Plasmids; D011773:Pyruvates / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011773:Pyruvates; D013439:Sulfhydryl Reagents; D010649:Phenylalanine; C017092:bromopyruvate; D004228:Dithionitrobenzoic Acid; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D003545:Cysteine