Molecular Mechanism of Photoregulation of a Plant Enzyme by Phosphorylation/Dephosphorylation

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Phosphoenolpyruvate carboxylase(PEPC)involved in C4 photosynthesis is known to be activated under light conditions and deactivated under dark conditions. This photoregulation is mediated by phosphorylation of PEPC. The study was undertaken to obtain a clue to the molecular mechanism for this process. Previously we found that PEPC of maize(a C4 plant)can be phosphorylated by a mammalian cyclic AMP-dependent protein kinase and is l e dto the activated state similarly to the activation in vivo. Furthermore, we identified the site of phosphorylation to be S e r - 1 4. A protein kinase which phosphorylates PEPC(PEPC-PK)was partially purified from maize green leaves and the site of phosphorylation was shown to be identical with the site phosphorylated by the mammalian protein kinase. A recombinant maize PEPC produced in E. coli cells, which is assumed to be free of phosphorylation was prepared and its kinetic properties were compared with the dark-form PEPC(40% phosphorylated)prepared from maize leaves. Kinetic propertied such as Km for PEP and Ki for malate were not significantly different from one another. To characterize PEPC-PK, a series of PK inhibitors with different specificities were tested and it was found that the enzyme was strongly inhibited by the inhibitor for Ca^<2+>- calmodulin-dependent PK such as myosin light chain kinase. Furthermore, the enzyme was inhibited by EGTA and the inhibition was relieved by the addition of Ca^<2+>. These results strongly suggest that the light signal is transduced to PK by elevating the intracellular concentration of Ca^<2+>. Molecular characterization of PK and cloning of its cDNA is now under progress.