| Project/Area Number |
10480157
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
IZUI Katsura Kyoto University, Graduate School of Biostudies, Professor, その他の独立研究科, 教授 (20025414)
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| Co-Investigator(Kenkyū-buntansha) |
KAI Yasushi Osaka University, Graduate School of Technology, Professor, 工学研究科, 教授 (40029236)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥9,700,000 (Direct Cost: ¥9,700,000)
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| Keywords | CO_2-fixation enzyme / phosphoenolpyruvate carboxylase / X-ray crystallography / protein engineering / site-directed mutagenesis / enzyme reaction mechanism / allosteric regulation mechanism / regulation by phosphorylation / 大腸菌 / トウモロコシ / ホスホエトルピルビン酸カルボキシラーゼ / C4光合成 / 部位特異的変異 |
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| 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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