Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies

María S. Orellana, Vasthi López, Elena Uribe, Marcia Fuentes, Mónica Salas, Nelson Carvajal

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn2+. The effect was reversed by hydroxylamine and involved a residue with a pKa of 6.5 ± 0.1. Half activation with Mn2+ was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in Km for arginine or Ki for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn2+ and contained < 0.1 Mn2+/subunit, whereas H101N was half active and contained 1.2 ± 0.1 Mn2+/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state.

Original languageEnglish
Pages (from-to)155-159
Number of pages5
JournalArchives of Biochemistry and Biophysics
Volume403
Issue number2
DOIs
Publication statusPublished - 15 Jul 2002
Externally publishedYes

Keywords

  • Arginase
  • Diethyl pyrocarbonate
  • Histidine residue
  • Human liver
  • Manganese
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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