Poster: Photosynthesis

Abs # 312: Enzymatic characterization of the recombinant phosphoenolpyruvate carboxylase kinase (PEPC-PK) from a C4 plant, Flaveria trinervia: Possible redox regulation

Presenter:	Izui, Katsura , izui@kais.kyoto-u.ac.jp
Authors	Izui, Katsura  (A)   Tsuchida, Yuhei  (A)   Agetsuma, Masakazu  (A)   Ohshima, Kenta  (A)   Furumoto, Tsuyoshi  (A)
Affiliations:	(A): Graduate School of Biostudies, Kyoto University

PEPC, a key enzyme in C4 photosynthesis, is controlled by reversible phosphorylation at a conserved Ser residue near the N-terminus. Recently, we cloned and characterized a cDNA for PEPC-PK from a C4 plant, Flaveria trinervia, which phosphorylates only PEPC (Tsuchida et al., FEBS Lett. 507,318-22 (2001)). On the other hand, we purified PEPC-PK from maize leaves and found that the enzyme is readily inactivated in vitro under non-reducing conditions and recovered by DTT or more efficiently by thioredoxin, suggesting possible redox regulation (Saze et al., Plant Cell Physiol. 42, 1295-1302 (2001)). Here.we report that the purified recombinant FtPEPC-PK was inactivated under mild oxidative conditions such as dialysis under air, treatment with oxidized glutathione (GSSG) or with low conc. of Cu²⁺. The activity could be recovered by DTT in a conc.-dependent manner. The formation of not inter- but intra-molecular disufide formation accompanied with inactivation was strongly indicated by the assay in which gel-mobility shift is correlated with the number of SH groups modifiable with a large-sized chemical modifier (AMS with an Mr of 536). The results of experiments to locate the two Cys residues involved in disufide formation among 4 candidates will be presented. The change of phosphorylation state of PEPC under oxidative stress will be discussed in terms of redox regulation of PEPC-PK.