Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAM plant Aptenia cordifolia

Two isoforms of NADP-dependent malic enzyme (NADP-ME) with the same molecular mass 72 kDa and different isoelectric points, 6.1 and 6.4, were found in crude extracts from the leaves of Aptenia cordifolia, a constitutive CAM plant. In the roots, only one isoform of 72 kDa was found, with a pi of 6.1. The isoform of pI 6.4 was partially purified from leaves to a final specific activity of 30.14 U mg(-1), a value similar to the photosynthetic isozymes. This enzyme showed a native mass of 264 kDa, suggesting a homotetramer. An optimal pH of 7.3 and K-m values for NADP and L-malate 13 muM and 1.1 mM, respectively, were determined. The enzymatic activities and the level of immunoreactive. protein did not vary with the day/night cycle. The enzyme was strongly and competitively inhibited by oxaloacetate (OAA), L-aspartate and phosphoenolpyruvate (PEP) and to a lesser degree by citrate, suggesting that NADP-ME activity might be subject to metabolite control. At night, high levels of OAA, L-aspartate and citrate might inhibit NADP-ME, avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation Of L-malate. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.