Loginova N V, Sokolov A P, Trotsenko Iu A
Biokhimiia. 1982 Oct;47(10):1629-34.
Pyruvate carboxylase of the facultative methylotroph Pseudomonas oleovorans was purified 40-fold by ammonium sulfate fractionation, gel filtration on Ultrogel AcA 34, ion-exchange chromatography on DEAE-Biogel A and concentration on DEAE-Sepharose CL-6B. The enzyme exerts its maximal activity in the presence of Mg2+ (pH 7.5, 40 degrees), is unstable and completely inactivated within 6 hrs at 25 degrees. In the presence of Mg2+ monovalent cations stimulate the enzyme activity. The molecular weight of pyruvate carboxylase as determined by gel filtration of Sepharose CL-6B is 300,000. The enzyme molecule contains biotin. The apparent Km values for pyruvate, ATP and HCO3- are 1.77, 0.19 and 0.23 mM, respectively. CoASAc, alpha-ketoglutarate and glutamate have no effect on the enzyme activity. The enzyme is inhibited by aspartate, malate, oxaloacetate and is activated by citrate, isocitrate and phosphosugars. The role of pyruvate carboxylase in methylotrophic metabolism of Ps. oleovorans is discussed.
通过硫酸铵分级沉淀、在Ultrogel AcA 34上进行凝胶过滤、在DEAE - Biogel A上进行离子交换色谱以及在DEAE - Sepharose CL - 6B上进行浓缩，将兼性甲基营养菌食油假单胞菌的丙酮酸羧化酶纯化了40倍。该酶在Mg2 +存在下（pH 7.5，40℃）发挥最大活性，不稳定，在25℃下6小时内完全失活。在Mg2 +存在下，单价阳离子刺激酶活性。通过Sepharose CL - 6B凝胶过滤测定的丙酮酸羧化酶分子量为300,000。酶分子含有生物素。丙酮酸、ATP和HCO3 - 的表观Km值分别为1.77、0.19和0.23 mM。CoASAc、α - 酮戊二酸和谷氨酸对酶活性无影响。该酶被天冬氨酸、苹果酸、草酰乙酸抑制，被柠檬酸、异柠檬酸和磷酸糖激活。讨论了丙酮酸羧化酶在食油假单胞菌甲基营养代谢中的作用。
