A CO_2 fixation enzme (PEP carboxylase) : Functional analysis by genetic engineering and X-ray chrystallography

磷酸烯醇式丙酮酸羧化酶（PEPC）是一种重要的CO_2固定酶。在C4植物中，该酶催化C4植物光合作用的初级CO_2同化反应。1)我们在国际上首次用X射线晶体学方法阐明了PEPC的三维结构，为基因工程的功能分析奠定了基础。2)从大肠杆菌PEPC与别构抑制剂天冬氨酸配体的结构出发，提出了一种独特的别构抑制机制模型。3)从E. coli PEPC与PEP类似物DCDP连接后，明确了催化位点的位置。4)对玉米PEPC进行了结晶，并对其三维结构进行了解析。通过比较E. coli和Zea mays的反应机理，推导出一个合理的反应机理模型。5)制备了在调节磷酸化位点附近具有突变的各种重组玉米酶，并且正在研究其与特定蛋白激酶的相互作用。6)通过晶体学分析确定了玉米PEPC的变构激活剂葡萄糖6-磷酸的作用位点，并通过定点突变鉴定了所涉及的氨基酸残基。7)一个柔性环桥连在催化位点上是催化活性不可或缺的，主要参与另一种底物HCO_3的结合。8)首次从C_4植物中克隆了一个与PEPC磷酸化调控有关的蛋白激酶的cDNA，并对其进行了鉴定。

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.