Abstract
Phosphoenolpyruvate carboxylase (PEPC), a key enzyme in C4 photosynthesis, is controlled by reversible phosphorylation at a conserved Ser residue near the N-terminus. Recently, we have cloned a PEPC kinase (PEPC-PK) from a C4 plant, Flaveria trinervia. Furthermore, we suggested that this PEPC-PK and purified PEPC-PK from maize leaves were redox-regulated. We now report the enzymatic characterization, especially redox-regulation of recombinant PEPC-PK.
Purified recombinant enzyme was inactivated by mild oxidation with dissolved oxygen, GSSG or Cu2+, but the activity could be recovered by DTT in a concentration-dependent manner. To assess the redox state of PEPC-PK, reduced and oxidized PEPC-PK modified with AMS (M.W=536.44), a SH-modifier were separated by non-reducing SDS-PAGE. Two bands corresponding to oxidized and reduced form PEPC-PK could be detected as a monomer, and the oxidized form was shifted to reduced form by DTT. These results suggested that PEPC-PK activity was controlled through an intramolecular thiol/disulfide formation.
