PMID:9169023
| Citation |
Giorgianni, F, Beranová, S, Wesdemiotis, C and Viola, RE (1997) Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli. Arch. Biochem. Biophys. 341:329-36 |
|---|---|
| Abstract |
Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-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 proteolytic 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. |
| Links |
PubMed Online version:10.1006/abbi.1997.9983 |
| Keywords |
Aspartate Ammonia-Lyase/antagonists & inhibitors; Aspartate Ammonia-Lyase/chemistry; Aspartate Ammonia-Lyase/genetics; Aspartic Acid/analogs & derivatives; Aspartic Acid/pharmacology; Bacterial Proteins/antagonists & inhibitors; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Chromatography, High Pressure Liquid; Cysteine/chemistry; Deamination; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Fumarates/metabolism; Fumarates/pharmacology; Lysine/chemistry; Mutagenesis, Site-Directed; Peptide Fragments/chemistry |
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