Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity
| Autor: | Bruno Sérgio do Amaral, Katia Celina S. Correa, Arlene G. Corrêa, Dulce Helena Ferreira de Souza, Quezia B. Cass |
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| Rok vydání: | 2019 |
| Předmět: |
chemistry.chemical_classification
biology GTP' Chemistry 010401 analytical chemistry PEPCK purification General Chemistry Metabolism Ligand (biochemistry) medicine.disease_cause 01 natural sciences 0104 chemical sciences PEPCK activity assay Enzyme Biochemistry biology.protein medicine Citrate synthase Phosphoenolpyruvate carboxykinase activity LC-MS/MS Phosphoenolpyruvate carboxykinase Escherichia coli |
| Zdroj: | Journal of the Brazilian Chemical Society v.30 n.10 2019 Journal of the Brazilian Chemical Society Sociedade Brasileira de Química (SBQ) instacron:SBQ Journal of the Brazilian Chemical Society, Volume: 30, Issue: 10, Pages: 2105-2113, Published: 21 OCT 2019 |
| ISSN: | 0103-5053 |
| Popis: | Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1). |
| Databáze: | OpenAIRE |
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