Molecular and Regulatory Properties of Leucoplast Pyruvate Kinase from Brassica napus (Rapeseed) Suspension Cells

Plastidic pyruvate kinase (PKp) from Brassica napus suspension cells was purified 431-fold to a final specific activity of 28 μmol phosphoenolpyruvate (PEP) utilized/min/mg protein. SDS–PAGE, immunoblot and gel filtration analyses indicated that this PKp exists as a 380-kDa heterohexamer composed of equal proportions of 64- (α-subunit) and 58-kDa (β-subunit) polypeptides. The N-terminal sequence of the PKp α- and β-subunits exhibited maximal identity with the corresponding regions deduced from putative PK genes of Arabidopsis thaliana and Methylobacterium extorquens, respectively. B. napus PKp displayed a sharp pH optimum of pH 8.0, and hyperbolic saturation kinetics with PEP and ADP (Km = 0.052 and 0.14 mM, respectively). 6-Phosphogluconate functioned as an activator (Ka = 0.12 mM) by increasing Vmax by approximately 35% while decreasing the Km(PEP) and Km(ADP) values by 40 and 50%, respectively. 2-Oxoglutarate and oxalate were the most effective inhibitors (I50 = 8.3 and 0.23 mM, respectively). A model is presented which highlights the role of 6-phosphogluconate in coordinating stromal NADPH and ATP production for anabolic processes of B. napus leucoplasts.