Elimination of the Sensitivity of L-Aspartase to Active-Site-Directed Inactivation without Alteration of Catalytic Activity

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

Research output: Contribution to journalArticle

9 Citations (Scopus)

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-β-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.

Original languageEnglish (US)
Pages (from-to)3529-3535
Number of pages7
JournalBiochemistry
Volume34
Issue number11
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

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Aspartate Ammonia-Lyase
Catalyst activity
Catalytic Domain
Enzymes
Escherichia coli
Cysteine
Viola
Sulfhydryl Reagents
Mutagenesis
Nucleophiles
Biochemistry
Sequence Homology
Site-Directed Mutagenesis
Catalysis
Aspartic Acid
Mass spectrometry
Mass Spectrometry
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Elimination of the Sensitivity of L-Aspartase to Active-Site-Directed Inactivation without Alteration of Catalytic Activity. / Giorgianni, Francesco; Beranova, Sarka; Wesdemiotis, Chrys; Viola, Ronald E.

In: Biochemistry, Vol. 34, No. 11, 01.01.1995, p. 3529-3535.

Research output: Contribution to journalArticle

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