Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: Kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

Ana L. Stern, Emmanuel Burgos, Laurent Salmon, Juan J. Cazzulo

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km, values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His 138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the knowledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

Original languageEnglish (US)
Pages (from-to)279-285
Number of pages7
JournalBiochemical Journal
Volume401
Issue number1
DOIs
StatePublished - Jan 1 2007
Externally publishedYes

Keywords

  • Chagas disease
  • Pentose phosphate pathway
  • Reaction mechanism
  • Ribose 5-phosphate isomerase (Rpi)
  • Trypanosoma cruzi
  • Type B ribose 5-phosphate isomerase (RpiB)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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