title: Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica.
abstract: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD⁺ or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: Veronika L
authors.initials: VL
authors.lastname: Zinsser
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Hoey
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Trudgett
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: d.timson@qub.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
mesh_terms: D000818:Animals; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005210:Fasciola hepatica / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005470:Fluorometry / Q000379:methods; D005986:Glyceraldehyde 3-Phosphate / Q000737:chemistry / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D005986:Glyceraldehyde 3-Phosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Drug target; G3PDH; Glycolytic enzyme; Neglected tropical disease; Trematode; Vaccine target