Phosphoglycerate mutase from Trypanosoma brucei is hyperactivated by cobalt in vitro, but not in vivo

Abstract

Production of ATP by the glycolytic pathway in the mammalian pathogenic stage of protists from the genus Trypanosoma is required for the survival of the parasites. Cofactor-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shows no similarity to the corresponding enzyme in humans, and has also been genetically validated as a target by RNAi experiments. It has previously been shown that trypanosomatid iPGAMs require Co(2+) to reach maximal activity, but the biologically relevant metal has remained unclear. In this paper the metal content in the cytosol of procyclic and bloodstream-form T. brucei (analysed by inductively coupled plasma-optical emission spectroscopy) shows that Mg(2+), Zn(2+) and Fe(2+) were the most abundant, whereas Co(2+) was below the limit of detection (<0.035 μM). The low concentration indicates that Co(2+) is unlikely to be the biologically relevant metal, but that instead, Mg(2+) and/or Zn(2+) may assume this role. Results from metal analysis of purified Leishmania mexicana iPGAM by inductively coupled plasma-mass spectrometry also show high concentrations of Mg(2+) and Zn(2+), and are consistent with this proposal. Our data suggest that in vivo cellular conditions lacking Co(2+) are unable to support the maximal activity of iPGAM, but instead maintain its activity at a relatively low level by using Mg(2+) and/or Zn(2+). The physiological significance of these observations is being pursued by structural, biochemical and biophysical studies.

Structural role of the active‐site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase

“…These parameters are consistent with those previously reported and within the range for iPGAMs from other organisms [13], indicating that the recombinant Tb iPGAM represents the natural enzyme. In our assays, the specific activity of Tb iPGAM at 37 °C in the presence of cobalt was approximately 3 thousand International Units (IU) mg −1 , which represents a sevenfold increase in enzymatic activity compared previously published results [2], probably due to different experimental conditions.…”

“…Two evolutionarily distinct classes of PGAM have been described: cofactor (2,3‐bisphosphoglycerate)‐dependent dPGAMs and cofactor‐independent iPGAMs [1]. iPGAMs are monomeric metalloenzymes with an absolute requirement for divalent metals that belong to the alkaline phosphatase superfamily and comprise a phosphatase and a transferase domain [2,3]. dPGAMs are usually dimeric or tetrameric, are not metalloenzymes belonging to the acid phosphatase superfamily.…”

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

“…A kinetic absorbance output was achieved using lactate dehydrogenase-mediated changes in NADH concentration through pyruvate conversion to lactate (Supplementary Fig. 2a, step 3a)1718. For the bioluminescent end point assay the ATP produced from the coupled enzymatic reactions is used by Firefly luciferase and luciferin to generate measurable light (Supplementary Fig.…”

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling

“…From the total magnesium content of T. brucei (35), a concentration of 6 m m magnesium in the cytosol of bloodstream cells could be estimated. This value fits to the 0.3–1.5 m m free magnesium reported for other cells taking into account that the total concentration is 10–20 times higher (36).…”