Nucleotide specificity of Saccharomyces

cerevisiae phosphoenolpyruvate
carboxykinase Kinetics, fluorescence
spectroscopy, and molecular
simulation studies
JOSÉ M. VILLARREAL, CLAUDIA BUENO, FELIPE ARENAS, ANA M.
JABALQUINTO, FERNANDO D. GONZÁLEZ-NILO, MARÍA V. ENCINAS, EMILIO
CARDEMIL,
Objetivo
 In this work, they have evaluated the kinetic and
thermodynamic interaction of purine and pyrimidine
nucleoside diphosphates and triphosphates with S.
cerevisiae PEP carboxykinase.
Phosphoenolpyruvate (PEP)
carboxykinases
 catalyze the nucleoside triphosphate-dependent reversible
decarboxylation of oxaloacetate (OAA) to yield PEP, CO2, and the
corresponding nucleoside diphosphate.

 PEP carboxykinases have an absolute requirement for divalent

cations for activity. One cation, preferentially a transition metal like
Mn2+
Materials and metods
 Recombinant S. cerevisiae PEP carboxykinase was purified
 Nucleotides and pyridoxal 5-phosphate (PLP) were purchased from
Sigma Chem. Co., St. Louis, USA.
 Oxaloacetate (OAA) was from Boehringer, Germany.
 All other reagents and auxiliary enzymes were of the highest purity
commercially available
 The phospho-pyridoxyl (P-pyridoxyl)-S. cerevisiae PEP
carboxykinase adduct was prepared with a labelling stoichiometry of
0.6–0.8 mole of P-pyridoxyl group per mole of enzyme subunit.