title: The phosphoglycerate kinase isoenzymes have distinct roles in the regulation of carbohydrate metabolism in Trypanosoma cruzi.
abstract: The glycolytic enzyme phosphoglycerate kinase (PGK) is present in Trypanosoma cruzi as three isoenzymes, two of them located inside glycosomes (PGKA and PGKC) and another one in the cytosol (PGKB). The three isoenzymes are expressed at all stages of the life cycle of the parasite. A heterologous expression system for PGKA (rPGKA) was developed and the substrate affinities of the natural and recombinant PGKA isoenzyme were determined. Km values measured for 3-phosphoglycerate (3PGA) were 174 and 850 μM, and for ATP 217 and 236 μM, for the natural and recombinant enzyme, respectively. No significant differences were found between the two forms of the enzyme. The rPGKA was inhibited by Suramin with Ki values of 10.08 μM and 12.11 μM for ATP and 3PGA, respectively, and the natural enzyme was inhibited at similar values. A site-directed mutant was created in which the 80 amino acids PGKA sequence, present as a distinctive insertion in the N-terminal domain, was deleted. This internally truncated PGKA showed the same Km values and specific activity as the full-length rPGKA. The natural PGKC isoenzyme was purified from epimastigotes and separated from PGKA through molecular exclusion chromatography and its kinetic characteristics were determined. The Km value obtained for 3PGA was 192 μM, and 10 μM for ATP. Contrary to PGKA, the activity of PGKC is tightly regulated by ATP (substrate inhibition) with a Ki of 270 μM, suggesting a role for this isoenzyme in regulating metabolic fluxes inside the glycosomes.
authors.affiliation: Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: Ximena
authors.initials: X
authors.lastname: Barros-Álvarez
authors.affiliation: Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: Ana J
authors.initials: AJ
authors.lastname: Cáceres
authors.affiliation: Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela; Research Unit for Tropical Diseases, de Duve Institute, Avenue Hippocrate 74, B-1200 Brussels, Belgium.
authors.forename: Paul A M
authors.initials: PA
authors.lastname: Michels
authors.affiliation: Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Concepción
authors.affiliation: Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela. Electronic address: wilqui@ula.ve.
authors.forename: Wilfredo
authors.initials: W
authors.lastname: Quiñones
mesh_terms: D000818:Animals; D015153:Blotting, Western; D050260:Carbohydrate Metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008018:Life Cycle Stages; D008830:Microbodies / Q000201:enzymology; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014344:Trypanocidal Agents / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D014344:Trypanocidal Agents; D013498:Suramin; D010735:Phosphoglycerate Kinase
keywords: Carbohydrate metabolism regulation; Glycolysis; Isoenzymes; Phosphoglycerate kinase; Substrate inhibition; Trypanosoma cruzi