| Research Abstract |
Phosphoenolpyruvate carboxylase (PEPC) is an important CO_2-fixation enzyme. In C4 plants, the enzyme catalyzes the primary CO_2 assimilation reaction for photosynthesis in C4 plants. 1) We elucidated the 3-dimensional structure of PEPC by X-ray crystallography for the first time in the world, and provided a basis for functional analysis by genetic engineering. 2) From the structure of E, coli PEPC liganded with an allosteric inhibitor, aspartate, a unique model for the allosteric inhibition mechanism was proposed. 3) From the structure of E. coli PEPC liganded with PEP analogue, DCDP, the location of catalytic site was unequivocally established. 4) PEPC of Zea mays was crystallized and its 3-dimensional structure was also elucidated. By comparing the structures of PEPCs from E. coli and Zea mays, a plausible model for the reaction mechanism was deduced. 5) Various recombmant maize enzymes having mutations near the regulatory phosphorylation site were prepared and its interaction with a specific protein kinase is under investigation. 6) The site of glucose 6-phosphate, an allosteric activator of maize PEPC, was suggested by crystallographic analysis and the amino acid residues involved were identified by site-directed mutagenesis. 7) A flexible loop bridging over the catalytic site was shown to be indispensable for catalytic avtivity and mainly involved in the binding of another substrate, HCO_3. 8) A cDNA for protein kinase involved in the regulatory phsphorylation of PEPC was cloned from a C4 plant for the first time and characterized.
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