Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.).
Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.).
复制标题
鹰嘴豆 (Cicer arietinum L.) 未成熟豆荚中磷酸烯醇丙酮酸羧化酶的纯化和特性。
DOI：
10.1104/pp.80.2.369
复制
发表时间：
1986
期刊：
影响因子：
7.4
通讯作者：
R. Singh
中科院分区：
文献类型：
--
作者：
H. R. Singal;R. Singh
通过硫酸铵分级、DEAE-纤维素层析和Sephadex G-200凝胶过滤，从鹰嘴豆未成熟的豆荚中纯化得到均一的磷酸烯醇式丙酮酸羧化酶(EC 4.1.1.31)，回收率约为29%。纯化的酶是由四个相同亚基组成的四聚体，在pH为8.1时酶活最高，相对分子质量约20万道尔顿。该酶的活性需要专一性的镁离子。该酶与磷酸烯醇式丙酮酸的反应符合典型的双曲线动力学，K(M)为0.74毫摩尔，而随着HCO(3)(-)浓度的增加，酶呈S型反应，S(0.5)为7.6毫摩尔。该酶被葡萄糖-6-磷酸、α-甘油磷酸、3-磷酸甘油酸和1，6-二磷酸果糖等无机磷酸和磷酸酯激活，被三磷酸核苷酸、有机酸和二价阳离子Ca(2+)、Mn(2+)抑制。草酰乙酸酯和苹果酸对该酶具有非竞争性抑制作用。葡萄糖-6-磷酸逆转草酰乙酸酯和苹果酸的抑制作用。
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.