title: Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica.
abstract: Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a β-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis-Menten kinetics in both directions, with Km values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s(-1) for the conversion of dihydroxyacetone phosphate and 1900 s(-1) for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. β-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.
authors: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors: Veronika L
authors: VL
authors: Zinsser
authors: Elizabeth M
authors: EM
authors: Hoey
authors: Alan
authors: A
authors: Trudgett
authors: David J
authors: DJ
authors: Timson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005210:Fasciola hepatica / Q000737:chemistry / Q000201:enzymology*; D005786:Gene Expression Regulation; D007700:Kinetics; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000737:chemistry; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
chemical_list: D010728:Phosphoenolpyruvate; D014305:Triose-Phosphate Isomerase
keywords: Dihydroxyacetone phosphate; Glyceraldehyde 3-phosphate; Neglected tropical disease; Trematode protein; β-Barrel protein