Kinetic Characterization of Recombinant PPi-dependent Phosphofructokinase from Orange

R.J. MUCHUT; C.V. PIATTONI; F.E. PODESTÁ; A.A. IGLESIAS

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

SAIB

Pyrophosphate-dependent phosphofructokinase (PPi-PFKase; EC 2.7.1.90) is a glycolytic enzyme present in higher plants, some bacteria and protozoa. The enzyme performs the reversible phosphorylation of fructose-6-phosphate (Fru6P) using PPi, as an alternative for the reaction catalyzed by the ATP-dependent enzyme (ATP-PFKase, EC 2.7.1.11). PPi-PFKase purified from plant extracts was found composed by two different subunits. In this work, we carried out the molecular cloning and expression of the genes encoding for and subunits of PPi-PFKase from Citrus sinensis. Both genes were expressed and co-expressed in Escherichia coli to obtain different active forms of the enzyme that were kinetically characterized. Fructose-2,6-bis-phosphate (Fru2,6bisP) activated the heterooligomeric PPi-PFKase by increasing Vmax from 40 U/mg to 90 U/mg and the affinity toward Fru6P and Mg2+ by ~7- and ~2-fold, respectively. The homo-oligomeric enzymes were 570-fold (homo- ) and 2350-fold (homo- ) less active than the hetero-oligomer. Fru2,6bisP showed no effect on the homo- enzyme and inhibited the homo- PPi-PFKase, mainly by decreasing ~21-fold the affinity toward Fru6P. Results suggest that plant PPi-PFKase could be finely regulated by an allosteric mechanism, and they also support the view that regulation could be complemented by the differential expression of the enzyme subunits.