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

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 (K (m) ) and K (A) 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 Angstrom resolution were obtained and will permit us to pursue the determination of structure.