PARENT SESSION
Posters P5B Light, redox and metabolic regulation: Dark Reactions. Abstracts (528-530)

Biochemical evidence of redox regulation of phosphoenolpyruvate carboxylase kinase. Tsuyoshi Furumoto^*,1, Yuhei Tsuchida¹, Ken Motohashi^2,3, Toru Hisabori^2,3, Katsura Izui¹, ¹ Graduate School of Biostudies, Kyoto University, Kyoto, Japan² Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan³ ATP System Project, ERAO, JST, Nagatsuta, Midori-ku, Yokohama, Japan

ABSTRACT- In C4 plants, phosphoenolpyruvate carboxylase (PEPC) catalyzes the CO2-fixation reaction in the C4 cycle in the cytoplasm of mesophyll cells. The activity of PEPC is not only regulated by allosteric effectors but also by reversible phosphorylation at its conserved Ser residue near the N-terminus. The phosphorylation causes the decrease of the sensitivity to feed-back inhibition by malate. Interestingly, there are found protein kinases whose substrate is confined to PEPC (PEPC-PK), though it consists solely of the core domain of PK (Nimmo et al. (2003) Arch. Biochem. Biophys. 414, 189-196; Izui et al. (2004) Annu. Rev. Plant Biol. 55, 69-84). Recently, we cloned a cDNA for PEPC-PK from a C4 plant Flaveria trinervia (Tsuchida et al. (2001) FEBS Lett. 507, 318-322). As we previously reported on purified maize PEPC-PK (Saze et al. (2001) Plant and Cell Physiol. 42, 1295-1302), the F. trinervia recombinant PEPC-PK showed the redox-sensitivity in vitro. Here, we show several lines of biochemical evidence for the redox-regulation. (1) PEPC-PK inactivated by oxidized form of glutathione (GSSG) could be reactivated completely either with low conc. of thioredoxin (Trx) (1 M Trx and 100 M DTT) or with Trx/NADPH/NADPH- dependent Trx reductase (NTR)-coupling system (1 M Trx, 100 M NADPH, 50 nM NTR). And a cytosolic type of Trx, Trx-h, was more effective than the chloroplast-isoforms, Trx-f and Trx-m. Glutaredoxin was far less effective. These results indicated that the oxidized PEPC-PK could be activated under naturally occurring mild reductive conditions, and that the reducing power could be directly transferred to PEPC-PK from Trx-h. (2) Among six Cys residues, a pairwise replacement of Cys53 and Cys250 to Ala, abolished the sensitivity to oxidants and reductants although every single mutant retained its sensitivity. From these results, the molecular basis of this regulation will be discussed.

KEY WORDS: Protein kinase, Thioredoxin, Redox, PEPC-PK