Ribose 5-phosphate Isomerase Type B from Trypanosoma Cruzi: Kinetic Properties and Site-directed Mutagenesis Reveal Information About the Reaction Mechanism.
From: Instituto de Investigaciones Biotecnologicas-Instituto Tecnologico de Chascomus (IIB-INTECH), Universidad Nacional de General San Martin-CONICET, Av General Paz 5445, INTI, edificio 24, 1650 San Martin, Buenos Aires, Argentina.
The Biochemical journal
- Publish Date: Jan 2007
- ISSN: 1470-8728
- Volume: 401
- Issue: 1
- Pages: 279-85
- Medium: Internet
- Language: English
- Citation (JAMA): Stern Ana L, Burgos Emmanuel, Salmon Laurent, et al. Ribose 5-phosphate Isomerase Type B from Trypanosoma Cruzi: Kinetic Properties and Site-directed Mutagenesis Reveal Information About the Reaction Mechanism.. Biochem. J. Jan 2007;401:279-85
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 His138, 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 know-ledge 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.
Mesh Headings (Keywords): Aldose-Ketose Isomerases, Amino Acid Sequence, Animals, Cloning, Molecular, Conserved Sequence, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Protozoan Proteins, Recombinant Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Trypanosoma cruzi
Check for Full Text / PubMed Unique Identifier (PMID): 16981853
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