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 act...
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Veröffentlicht in: | The Journal of biological chemistry 1992-03, Vol.267 (9), p.5762-5767 |
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Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)42618-X |