title: Elimination of the sensitivity of L-aspartase to active-site-directed inactivation without alteration of catalytic activity.
abstract: The catalytic activity of the enzyme L-aspartase from Escherichia coli has previously been shown to be sensitive to sulfhydryl reagents. The use of group-specific reagents, and a sequence homology comparison study among the fumarase-aspartase family of enzymes, has not, however, lead to the identification of a specific, essential cysteinyl residue. We have recently shown that L-aspartate-beta-semialdehyde is an alternative substrate for L-aspartase, producing fumaric acid semialdehyde (FAA) which specifically inactivates the enzyme [Schindler, J. F., & Viola, R. E. (1994) Biochemistry 33, 9365]. Proteolytic digests of the resulting inactivated enzyme have now been mapped by HPLC and mass spectrometry. A specific residue (Cys-273) has been determined to be the site of FAA modification. Site-directed mutagenesis of this cysteine in the E. coli enzyme has produced altered enzymes which are considerably less sensitive to active-site-directed inactivation, while retaining full catalytic activity. Thus, cysteine-273 has been identified as an active-site nucleophile that, while not directly involved in catalysis in L-aspartase, is poised to attack an activated double bond in an enzyme-bound product analogue.
authors.affiliation: Department of Chemistry, University of Akron, Ohio 44325-3601.
authors.forename: F
authors.initials: F
authors.lastname: Giorgianni
authors.forename: S
authors.initials: S
authors.lastname: Beranová
authors.forename: C
authors.initials: C
authors.lastname: Wesdemiotis
authors.forename: R E
authors.initials: RE
authors.lastname: Viola
mesh_terms: D000595:Amino Acid Sequence; D001220:Aspartate Ammonia-Lyase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping
chemical_list: D001220:Aspartate Ammonia-Lyase; D003545:Cysteine