Mechanistic analysis of the argE-encoded N-acetylornithine deacetylase

Farah Javid-Majd, John S. Blanchard

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Abstract

The E. coli argE-encoded N-acetyl-L-ornithine deacetylase has been cloned, expressed, and purified in high yield. The substrate specificity of the enzyme is relatively broad, with a number of α-N-acyl-L-amino acids exhibiting activity, including both α-N-acetyl- and α-N-formylmethionine that exhibit higher activity than α-N-acetyl-L-ornithine. Sequence homology suggests that the enzyme is a member of the metal-dependent aminoacylase family, and the purified enzyme contains a single atom of zinc per monomer. The activity of this enzyme can be increased greater than 2-fold by the addition of zinc, or 8-fold by the addition of cobalt. This suggests that the enzyme can accommodate two metal ions at the active site. The pH dependence of the kinetic parameters has been determined and revealed the presence of two enzymic groups, one functioning as a general base and one functioning as a general acid. Solvent kinetic isotope effects on the hydrolysis of N- acetylornithine have been determined, and a linear proton inventory suggests that a single proton transfer occurs in a partially rate-limiting step. A chemical mechanism is proposed and compared with other mechanisms determined for other members of the aminoacylase family.

Original languageEnglish (US)
Pages (from-to)1285-1293
Number of pages9
JournalBiochemistry
Volume39
Issue number6
DOIs
Publication statusPublished - Feb 15 2000

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ASJC Scopus subject areas

  • Biochemistry

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