Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity

Bruno Do Amaral,Quezia Cass,Arlene Corrêa,Dulce De Souza,Katia Celina Correa

doi:10.21577/0103-5053.20190079

Abstract

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).

Highlights

Phosphoenolpyruvate carboxykinase (PEPCK) is a widely distributed enzyme present in most organisms.[1]
Its catalytic activity is related to reversible decarboxylation and phosphorylation of oxaloacetate (OAA) to yield phosphoenolpyruvate (PEP) and carbon dioxide, in the presence of a nucleotide (adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP)) and has an absolute requirement for divalent metal ions as cofactor
PEPCK is involved in glycogenesis, while in Trypanosoma cruzi, the causative agent of Chagas’ disease, other trypanosomatids and Leishmania are involved in glucose catabolism.[5,6]

Summary

Introduction

Phosphoenolpyruvate carboxykinase (PEPCK) is a widely distributed enzyme present in most organisms.[1]. Based on the nucleotide specificity, PEPCKs can be divided into two families, whereby ATP-dependent (EC 4.1.1.49) are found in bacteria, yeasts, higher plants and trypanosomatids; and GTP-dependent (EC 4.1.1.32), in mollusks, insects, fungi and vertebrate animals.[1,2,3] Different levels of sequence homology are observed among PEPCKs from the same family, but a low identity (18-20%) is verified between PEPCKs from different families. PEPCK is involved in glycogenesis, while in Trypanosoma cruzi, the causative agent of Chagas’ disease, other trypanosomatids and Leishmania are involved in glucose catabolism.[5,6] The aerobic fermentation of glucose, in T. cruzi, leads to the production of reduced catabolites: succinate and L-alanine as major products linked to the re-oxidation of glycolytic NADH (reduced nicotinamide adenine dinucleotide). The production of succinate depends on CO2 fixation, which is mediated by PEPCK by the production of OAA from PEP in the glycosome. Glycosomal malate following entry into the cytosol can participate of the glutamate dehydrogenase pathway, leading to the production of L-alanine.[7,8]

Methods

Results

Conclusion