Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. a role for Glu274 in binding and correct positioning of the substrate guanidinium group

Nelson Carvajal, María S. Orellana, Mónica Salas, Paula Enríquez, Ricardo Alarcón, Elena Uribe, Vasthi López

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group was investigated by kinetic and site-directed mutagenesis studies. Putrescine and guanidinium ions (Gdn +) were slope-linear, competitive inhibitors with respect to agmatine and their bindings to the enzyme were not mutually exclusive. By site-directed mutagenesis, the E274A variant exhibiting about 1-2% of wild-type activity was obtained. Mutation produced a moderate, but significant, increase in the K m value for agmatine (from 1.1 ± 0.2 mM to 6.3 ± 0.3 mM) and the K i value for competitive inhibition by Gdn + (from 15.0 ± 0.1 mM to 44.2 ± 2.1 mM), but the K i value for putrescine inhibition (2.8 ± 0.2 mM) was not altered. The tryptophan fluorescence properties (λ max = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 → Ala mutation. The dimeric structure of the enzyme was also maintained. We conclude that Glu274 is involved in binding and positioning of the guanidinium moiety of the substrate for efficient catalysis. A kinetic mechanism involving rapid equilibrium random release of products is proposed for E. coli agmatinase.

Original languageEnglish
Pages (from-to)185-190
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume430
Issue number2
DOIs
Publication statusPublished - 15 Oct 2004
Externally publishedYes

Keywords

  • Agmatinase
  • E. coli
  • Glu274
  • Kinetic mechanism
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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