Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity

Autor(es)

do Amaral, Bruno S. ^[1] ; Correa, Katia Celina S. ^[1] ; Correa, Arlene G. ^[1] ; de Souza, Dulce H. F. ^[1] ; Cass, Quezia B. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Dept Quim, Rod Washington Luis Km 235, BR-13565905 Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of the Brazilian Chemical Society; v. 30, n. 10, SI, p. 2105-2113, OCT 2019.
• Citações Web of Science: 0

Resumo

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 (K-Mapp) values of 96 +/- 4 and 275 +/- 18 mu mol 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 mu mol L-1). (AU)

• Processo FAPESP: 14/50249-8 - Green chemistry: sustainable synthetic methods employing benign solvents, safer reagents, and bio-renewable feedstock
• Beneficiário: Arlene Gonçalves Corrêa
• Modalidade de apoio: Auxílio à Pesquisa - Programa Centros de Pesquisa em Engenharia
• Processo FAPESP: 13/01710-1 - Ligantes enzimáticos: novos modelos de triagem
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Temático