Flagellate protozoa from the order Trypanosomatida have developed a range of strategies to survive in their mammalian hosts. A consequence is that the glycolytic pathway has assumed an important role, especially in bloodstream-form Trypanosoma brucei, where it is essential as the sole producer of ATP. The seventh enzyme in the pathway, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shares no common properties with the corresponding enzyme in humans. This enzyme catalyses the conversion of 3PGA to 2PGA, with the requirement for metal ions to assist the catalytic function. In this study, two important biochemical and structural aspects of the enzyme were investigated: i) The in vitro and in vivo requirements for biologically relevant metal ions to support the activity of iPGAM, and ii) The ability of trypanosomatid iPGAM to exist in multiple conformations and oligomeric states in solution. The maximum activity of iPGAM in vitro requires Co2+, but this cannot be the case in vivo where ICP-OES analyses confirmed that Co2+ was essentially undetectable in T. brucei cytosolic fractions. The activity of iPGAM in vivo is therefore one of the lowest among the glycolytic enzymes. By contrast, Mg2+ and Zn2+ were found to be the most abundant metals in both cytosolic fractions and in purified bacterially expressed iPGAM. Our newly-developed multimode-plate reader discontinuous assay further revealed that of the biologically relevant metals, only Mg2+ can support iPGAM activity, but at less than 50% of the level of Co2+. By contrast, Zn2+ strongly inhibits iPGAM. This assay which was developed with minimal metal interference on the coupling enzymes, also showed that in solution, the ratio of the concentrations of 3PGA:2PGA (substrate:product) at equilibrium is not 1:1 as observed in the crystal structure, but is in fact 12:1, which may be due to the tighter binding of 2PGA to the enzyme. A series of biophysical analyses, notably by SEC-MALS showed that iPGAM from Leishmania mexicana, another trypanosomatid protozoan parasite exists in different forms and oligomeric states in solution, either as the closed-form monomer, openiii form monomer, or closed/open-form dimer which can be successfully separated by ion-exchange chromatography. The open-form LmiPGAM is particularly relevant for drug development, as the catalytic site in the closed-form structure is poorly inaccessible. Both virtual and high-throughput screening approaches were used to identify novel potential inhibitors. Out of a collection of 11 compounds tested at 1 mM, two showed substantial inhibition with 49% and 14% remaining activity. Taken together, the findings from this study demonstrated the potential of iPGAM to be a key modulator in controlling glycolytic flux in trypanosomes, and thus further validated it as an important drug target.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563726 |
| Date | January 2012 |
| Creators | Fuad, Fazia Adyani Ahmad |
| Contributors | Walkinshaw, Malcolm. : Gilmore, Linda |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/6237 |
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