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Biochem Biophys Rep. 2016 Jun 8;7:124-129. doi: 10.1016/j.bbrep.2016.06.007. eCollection 2016 Sep.

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase.

Author information

1
Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), BIFI-IQFR (CSIC) Joint Unit, Universidad de Zaragoza, 50009 Zaragoza, Aragón, Spain.
2
Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
3
Fundación ARAID, Gobierno de Aragón, Zaragoza, Spain.
4
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
5
IIS Aragón, 50009 Zaragoza, Spain.

Abstract

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn2+ ions for activity; Mg2+ ions reduce the Km for Mn2+ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (kcat=2.7 s-1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.

KEYWORDS:

Gluconeogenesis; Glyceroneogenesis; Human mitochondrial phosphoenolpyruvate carboxykinase (PCK); Kinetics; Purification

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