Singal H R, Singh R
Department of Chemistry and Biochemistry, Haryana Agricultural University, Hisar-125004, India.
Plant Physiol. 1986 Feb;80(2):369-73. doi: 10.1104/pp.80.2.369.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg(2+) ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K(m) of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO(3) (-) with S(0.5) value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, alpha-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca(2+) and Mn(2+). Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.
磷酸烯醇式丙酮酸羧化酶（EC 4.1.1.31）通过硫酸铵分级分离、DEAE - 纤维素色谱以及经Sephadex G - 200的凝胶过滤，从鹰嘴豆未成熟豆荚中纯化至均一，回收率约为29%。纯化后的酶分子量约为200,000道尔顿，是由四个相同亚基组成的四聚体，在pH 8.1时表现出最大活性。酶活性特别需要Mg(2+)离子。该酶对磷酸烯醇式丙酮酸呈现典型的双曲线动力学，K(m)为0.74毫摩尔，而随着HCO(3) (-)浓度增加观察到S形响应，S(0.5)值为7.6毫摩尔。该酶被无机磷酸盐和磷酸酯如葡萄糖 - 6 - 磷酸、α - 甘油磷酸、3 - 磷酸甘油酸和果糖 - 1,6 - 二磷酸激活，被三磷酸核苷酸、有机酸以及二价阳离子Ca(2+)和Mn(2+)抑制。草酰乙酸和苹果酸非竞争性抑制该酶。葡萄糖 - 6 - 磷酸可逆转草酰乙酸和苹果酸的抑制作用。
