Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli

Francesco Giorgianni, Sarka Beranova, Chrys Wesdemiotis, Ronald E. Viola

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate β-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F. Giorgianni et al. (1995) Biochemistry 34, 3529). Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme- FAA derivative(s). HPLC and mass spectrometric analyses of pro-teolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide. The inactivation at this additional site involves a crosslink between cysteine- 140 and an adjacent lysine. Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity. The adjacent lysine-139 that is modified does appear to play a role in substrate binding. A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs.

Original languageEnglish (US)
Pages (from-to)329-336
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume341
Issue number2
DOIs
StatePublished - May 15 1997

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Aspartate Ammonia-Lyase
Escherichia coli
Cysteine
Aldehydes
Lysine
Substrates
Enzymes
Mutagenesis
Nucleophiles
Biochemistry
Enzyme activity
Site-Directed Mutagenesis
High Pressure Liquid Chromatography
Derivatives
Peptides

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli. / Giorgianni, Francesco; Beranova, Sarka; Wesdemiotis, Chrys; Viola, Ronald E.

In: Archives of Biochemistry and Biophysics, Vol. 341, No. 2, 15.05.1997, p. 329-336.

Research output: Contribution to journalArticle

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