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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

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 Kinase¹

Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes Number 6, 41012 Seville, Spain (R.A., S.G.M.-M., A.-B.F., C.E.); and Institut de Biotechnologie des Plantes, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 8618, Bâhtiment 630, Université de Paris-Sud, Centre d'Orsay cedex, France (J.V.)

Higher plant phosphoenolpyruvate 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 (C₄ PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C₄ 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) C₄ 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 Ca²⁺-independent PEPCk (50% inhibition of initial activity = 230 µM). This effect was highly specific because peptide C19 did not alter C₄ PEPC phosphorylation by either a partially purified sorghum leaf Ca²⁺-dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca²⁺-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 C₄ 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 C₄ 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.

¹ This work was supported by the Dirección General de Investigación Cientifica y Técnica (grant nos. PB97–0745–CO2–02 and BCM2001–2366–CO3–02), by the "Acción Integrada HispanoFrancesa HF2000–0009," and by the "Grupo de Investigación de Fisiología Vegetal CVI134" from La Junta de Andalucía.

^* Corresponding author; e-mail echeva{at}us.es; fax 34–954615780.

Received March 21, 2003; returned for revision March 25, 2003; accepted March 25, 2003.