Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli.
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.
Giorgianni
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F
Department of Chemistry, University of Akron, Ohio 44325-3601, USA.
Beranová
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S
Wesdemiotis
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Viola
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D001220:Aspartate Ammonia-Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003641:Deamination; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005650:Fumarates / Q000378:metabolism / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005650:Fumarates; D010446:Peptide Fragments; C106376:fumaric acid semialdehyde ethyl ester; D001224:Aspartic Acid; C006644:aspartic semialdehyde; C032005:fumaric acid; D001220:Aspartate Ammonia-Lyase; D008239:Lysine; D003545:Cysteine