Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase

Abstract

In this article, we report the results of an analysis of the glycolytic enzyme enolase (2-phospho-d-glycerate hydrolase) of Trypanosoma brucei. Enolase activity was detected in both bloodstream-form and procyclic insect-stage trypanosomes, although a 4.5-fold lower specific activity was found in the cultured procyclic homogenate. Subcellular localization analysis showed that the enzyme is only present in the cytosol. The T. brucei enolase was expressed in Escherichia coli and purified to homogeneity. The kinetic properties of the bacterially expressed enzyme showed strong similarity to those values found for the natural T. brucei enolase present in a cytosolic cell fraction, indicating a proper folding of the enzyme in E. coli. The kinetic properties of T. brucei enolase were also studied in comparison with enolase from rabbit muscle and Saccharomyces cerevisiae. Functionally, similarities were found to exist between the three enzymes: the Michaelis constant (Km) and KA values for the substrates and Mg2+ are very similar. Differences in pH optima for activity, inhibition by excess Mg2+ and susceptibilities to monovalent ions showed that the T. brucei enolase behaves more like the yeast enzyme. Alignment of the amino acid sequences of T. brucei enolase and other eukaryotic and prokaryotic enolases showed that most residues involved in the binding of its ligands are well conserved. Structure modelling of the T. brucei enzyme using the available S. cerevisiae structures as templates indicated that there are some atypical residues (one Lys and two Cys) close to the T. brucei active site. As these residues are absent from the human host enolase and are therefore potentially interesting for drug design, we initiated attempts to determine the three-dimensional structure. T. brucei enolase crystals diffracting at 2.3 A resolution were obtained and will permit us to pursue the determination of structure.

Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei

“…First, the model was optimized and extended with recent information about the kinetics of enzymes (25)(26)(27)(28). Moreover, it was modified on the basis of activities of enzymes as measured in lysates of in vitro cultured growing bloodstream form trypanosomes.…”

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

“…Glycolysis -The enzymes of the glycolytic pathway in trypanosomatids are organised in two cellular compartments, while in higher eukaryotes these enzymes are present in the cytoplasm. In T. brucei, the first seven enzymes of the pathway are present in the glycosome and transform glucose into 3-phosphoglycerate, while the three other enzymes are localised in the cytoplasm (Hannaert et al 2003, Bringaud et al 2006. T. brucei is an exclusively extracellular parasite and its metabolism is directly related to the concentration of nutrients in the medium.…”

“…The slender bloodstream forms of the parasite produce energy almost exclusively from glucose. The phosphoglycerol kinase localisation is either glycosomal or cytosolic, as observed in the promastigotes of different Leishmania species and in T. cruzi epimastigotes and are required to maintain the glycosomal ATP/ADP balance, which offers a rationale for the presence of both phosphoglycerate mutase and enolase in the cytosol (Hannaert et al 2003).…”

“…cruzi and Leishmania species require a more complex energy metabolic system and additional pathways, such as the Krebs cycle and oxidative phosphorylation, are compartmentalised in the mitochondria (Taylor & Gutteridge 1987, Hannaert et al 2003. In T. cruzi, the first enzyme of the pathway, a hexokinase (HK), was described as having different kinetic characteristics than the human enzyme.…”

“…The parasitic forms of trypanosomatids also have metabolic differences during their life cycle, such as the Krebs cycle in Trypanosoma brucei, which is nearly inactive in bloodstream forms found in the vertebrate host, but is active in the procyclic form in the invertebrate host (Hannaert et al 2003).…”