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.
Stephens
C M
CM
Department of Biology, University of Virginia, Charlottesville 22901.
Bauerle
R
R
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