A Conserved 19-Amino Acid Synthetic Peptide from the Carboxy Terminus of Phosphoenolpyruvate Carboxylase Inhibits the in Vitro Phosphorylation of the Enzyme by the Calcium-Independent Phosphoenolpyruvate Carboxylase Kinase

Higher plant phospho enol pyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein. Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C 4 PEPC) suggested that the interaction of phospho enol pyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C 4 PEPC interactions take place. In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum ( Sorghum vulgare Pers cv Tamaran) C 4 PEPC with PEPCk. In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca 2+ -independent PEPCk (50% inhibition of initial activity = 230 μm). This effect was highly specific because peptide C19 did not alter C 4 PEPC phosphorylation by either a partially purified sorghum leaf Ca 2+ -dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca 2+ -independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column. Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C 4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect. Specific antibodies raised against peptide C19 revealed that native C 4 PEPC could be in two different conformational states. The results are discussed in relation with the reported crystal structure of the bacterial ( Escherichia coli ) and plant (maize [ Zea mays ]) enzymes.