Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays:: Comparison with the C4-form enzyme
Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays:: Comparison with the C4-form enzyme
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DOI：
10.1093/oxfordjournals.pcp.a029446
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发表时间：
1998-08-01
影响因子：
4.9
通讯作者：
Izui, K
中科院分区：
文献类型：
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作者：
Dong, LY;Masuda, T;Izui, K
分离出玉米根型磷酸烯醇丙酮酸羧化酶 (PEPC) 的全长 cDNA。在编码区，根型 PEPC 在核苷酸水平上与玉米的 C-4 型和 C-3 型 PEPC 分别显示出 76% 和 77% 的同一性。在氨基酸水平上，根型与 C-4 型和 C-3 型 PEPC 分别有 81% 和 85% 的同一性。将整个编码区插入pET32a表达载体中，使其在T7启动子的控制下表达。纯化的重组根型PEPC在pH 8.0下的V-max约为28μmol min(-1)(mg蛋白质)(-1)。在pH 7.3或8.0下测定时，根型PEPC对PEP和Mg2+的K-m值是C-4型PEPC的十分之一或更少，而HCO3-的值约为C-4-型PEPC的二分之一。 pH 8.0的PEPC、6-磷酸葡萄糖和甘氨酸对pH 7.3的根型PEPC影响不大；它们引起 C-4 型 PEPC 的两倍激活，根型和 C-4 型 PEPC 在 pH 7.3 下的 K-i（L-苹果酸）值分别为 0.12 和 0.43 mM。玉米 PEPC 同工型之间的亲水特性比较表明，几段氨基酸序列可能以某种方式对其特征动力学特性做出贡献。根型PEPC被哺乳动物cAMP依赖性蛋白激酶和玉米叶蛋白激酶磷酸化，并且磷酸化酶对L-苹果酸不太敏感。
A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase (PEPC) was isolated. In the coding region, the root-form PEPC showed 76 and 77% identity with the C-4- and C-3-form PEPCs of maize, respectively, at the nucleotide level. At the amino acid level, the root-form was 81 and 85% identical to the C-4- and C-3-form PEPCs, respectively. The entire coding region was inserted into a pET32a expression vector so that it was expressed under the control of T7 promoter. The purified recombinant root-form PEPC had a V-max value of about 28 mu mol min(-1) (mg protein)(-1) at pH 8.0, The K-m values of root-form PEPC for PEP and Mg2+ were one-tenth or less of those of C-4-form PEPC when assayed at either pH 7.3 or 8.0, while the value for HCO3- was about one-half of that of C-4- form PEPC at pH 8.0, Glucose 6-phosphate and glycine had little effect on the root-form PEPC at pH 7.3; they caused two-fold activation of the C-4-form PEPC, The K-i (L-malate) values at pH 7.3 were 0.12 and 0.43 mM for the root- and C-4-form PEPCs, respectively. Comparison of hydropathy profiles among the maize PEPC isoforms suggested that several stretches of amino acid sequences may contribute in some way to their characteristic kinetic properties. The root-form PEPC was phosphorylated by both mammalian cAMP-dependent protein kinase and maize leaf protein kinase, and the phosphorylated enzyme was less sensitive to L-malate.