Chen Y, Wurtele E S, Wang X, Nikolau B J
Department of Food Science, Iowa State University, Ames, Iowa 50011.
Arch Biochem Biophys. 1993 Aug 15;305(1):103-9. doi: 10.1006/abbi.1993.1398.
3-Methylcrotonyl-CoA carboxylase, a biotin enzyme, was purified from embryos of Daucus carota. Polyethylene glycol precipitation and monomeric avidin affinity chromatography were used to purify all biotin enzymes from cell-free extracts of embryos. The resulting 3-methylcrotonyl-CoA carboxylase preparation had a specific activity of 745 nmol/min.mg protein, representing a 3725-fold purification of the enzyme and a 135% recovery of activity. Fractionation of the purified biotin-containing proteins by anionic exchange chromatography using Q-Sepharose partially resolved the 3-methylcrotonyl-CoA carboxylase from the other biotin enzymes. 3-Methylcrotonyl-CoA carboxylase has a biotin-containing subunit with a molecular mass of about 78,000 Da and a non-biotin-containing subunit of about 65,000 Da. The native enzyme is 987,000 Da. The optimum pH for activity is between 8.0 and 8.4. The apparent Km values for the substrates 3-methylcrotonyl-CoA, sodium bicarbonate, and ATP are 42 +/- 2 microM, 4.0 +/- 0.9 mM, and 21 +/- 2 microM, respectively. The enzyme is inhibited by acetoacetyl-CoA and palmitoyl-CoA.
3-甲基巴豆酰辅酶A羧化酶是一种生物素酶，从胡萝卜胚中纯化得到。采用聚乙二醇沉淀法和单体抗生物素蛋白亲和色谱法从胚的无细胞提取物中纯化所有生物素酶。所得的3-甲基巴豆酰辅酶A羧化酶制剂的比活性为745 nmol/分钟·毫克蛋白，该酶的纯化倍数为3725倍，活性回收率为135%。使用Q-琼脂糖通过阴离子交换色谱法对纯化的含生物素蛋白进行分级分离，可将3-甲基巴豆酰辅酶A羧化酶与其他生物素酶部分分离。3-甲基巴豆酰辅酶A羧化酶有一个含生物素的亚基，分子量约为78,000 Da，还有一个不含生物素的亚基，分子量约为65,000 Da。天然酶的分子量为987,000 Da。该酶的最适pH值在8.0至8.4之间。底物3-甲基巴豆酰辅酶A、碳酸氢钠和ATP的表观Km值分别为42±2 μM、4.0±0.9 mM和21±2 μM。该酶受到乙酰乙酰辅酶A和棕榈酰辅酶A的抑制。
