Abstract

A family of synthetic peptides modelled after the highly conserved, N-terminal phosphorylation domain of C₄ phosphoenolpyruvate carboxylase (PEPC) and a complementary set of recombinant mutant target proteins were exploited to investigate the local structural requirements for phosphorylation of this cytosolic C₄ enzyme by its Ca²⁺-independent protein kinase. The only peptide homolog examined that was significantly phosphorylated by maize (Zea mays L.) leaf PEPC-kinase spanned the P−5 through P+16 region surrounding the target serine residue in maize PEPC (fifth through sixteenth residues on the N- and C-terminal sides, respectively, of the phosphorylatable serine at position ‘P’). However, its apparent K_m value was 200-times that of intact C₄ PEPC. The results from the related site-directed mutagenesis experiments with the recombinant sorghum (Sorghum vulgare) C₄ -enzyme indicated that alteration of several highly conserved residues flanking the target serine with non-conservative Ala substitutions at the P−4, P−3, and P+10 positions had only modest effects, if any, on its in-vitro phosphorylation by maize PEPC-kinase. These collective findings implicate a secondary site(s) of interaction in C₄ PEPC, removed from the N-terminal phosphorylation domain, that is also an important recognition element for its low abundance, highly regulated protein-serine/threonine kinase.