Cloning, Expression, and Characterization of a Root-Form Phosphoenolpyruvate Carboxylase from Zea mays: Comparison with the C4-Form Enzyme |
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Authors: | Dong Long-Ying; Masuda Takao; Kawamura Takao; Hata Shingo; Izui Katsura |
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Institution: | Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University Sakyo-ku, Kyoto, 606-8502 Japan |
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Abstract: | A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase(PEPC) was isolated. In the coding region, the root-form PEPCshowed 76 and 77% identity with the C4- and C3-form PEPCs ofmaize, respectively, at the nucleotide level. At the amino acidlevel, the root-form was 81 and 85% identical to the C4- andC3-form PEPCs, respectively. The entire coding region was insertedinto a pET32a expression vector so that it was expressed underthe control of T7 promoter. The purified recombinant root-formPEPC had a Vmax value of about 28 mol min1(mg protein)1at pH 8.0. The Km values of root-form PEPC for PEP and Mg2+were one-tenth or less of those of C4-form PEPC when assayedat either pH 7.3 or 8.0, while the value for HCO3 wasabout one-half of that of C4-form PEPC at pH 8.0. Glucose 6-phosphateand glycine had little effect on the root-form PEPC at pH 7.3;they caused two-fold activation of the C4-form PEPC. The Ki(L-malate) values at pH 7.3 were 0.12 and 0.43 raM for the root-and C4-form PEPCs, respectively. Comparison of hydropathy profilesamong the maize PEPC isoforms suggested that several stretchesof amino acid sequences may contribute in some way to theircharacteristic kinetic properties. The root-form PEPC was phosphorylatedby both mammalian cAMP-dependent protein kinase and maize leafprotein kinase, and the phosphorylated enzyme was less sensitiveto L-malate.
1These authors contributed equally to this work.
2Present address: Otsuka Chemical Co. Ltd., 463 Kagasuno, Kawauchi-cho,Tokushima, 771-0130 Japan.
3Present address: Sumitomo Pharmaceuticals Research Center,1-98, Kasugade, Naka 3-cho-me, Konohana-ku, Osaka, 554-0022Japan. |
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