Light/dark regulation of maize (Zea mays L.) leaf phosphoenolpyruvate carboxylase activity by reversible phosphorylation of serine-15

Cytoplasmic C$\sb4$ phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is reversibly activated by light. Evidence was presented to show that the molecular mechanism whereby PEPC is light/dark regulated is reversible phosphorylation of Ser-15 of this target enzyme from maize leaves. (1) An in vitro reconstituted phosphorylation system comprised of purified dark-form maize leaf PEPC, a protein kinase preparation partially purified from light-adapted maize leaves, and ATP$\cdot$Mg was developed to critically assess the effects of phosphorylation on the catalytic activity and malate sensitivity of PEPC. The results demonstrated that the protein kinase(s)-mediated changes in catalytic activity and malate sensitivity of the target enzyme are correlated with concomitant increases in its seryl-phosphorylation status, suggesting that phosphorylation of a seryl residue(s) is responsible for the light-activation of C$\sb4$ PEPC. (2) Purified dark-form maize leaf PEPC was $\sp{32}$P-phosphorylated/activated in vitro by the above reconstituted phosphorylation system. Automated Edman degradation analysis of the $\sp{32}$P-labeled phosphopeptide isolated from the tryptic digest of $\sp{32}$P-PEPC shows that a single serine residue is phosphorylated in vitro. The amino acid sequence of this regulatory phosphopeptide is His-His-Ser(P)-Ile-Asp-Ala-Gln-Leu-Arg. This nonapeptide corresponds exactly to residues 13 to 21 in the deduced primary structure of maize leaf PEPC. (3) Detached maize leaf tissue was fed with $\sp{32}$Pi and the in vivo $\sp{32}$P-labeled PEPC was then purified from light- and dark-adapted leaf tissue. Comparative sequence analysis of the $\sp{32}$P-phosphopeptides isolated from both light- and dark-forms of $\sp{32}$P-labeled PEPC established that (i) the in vivo phosphorylation site is the same as that identified in vitro, that is, Ser-15, and (ii) the degree of phsophorylation of Ser-15 in vivo is much greater in the light than in the dark