Alonso M J, De Arriaga D, Soler J
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Biochim Biophys Acta. 1988 Jul 20;955(2):175-86. doi: 10.1016/0167-4838(88)90191-4.
A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.
已从布氏梨形孢（Phycomyces blakesleeanus）NRRL 1555 (-) 的菌丝体中纯化出一种氨甲酰磷酸合酶。通过凝胶过滤法估计该酶的分子量为188,000。在十二烷基硫酸钠存在下进行的聚丙烯酰胺凝胶电泳显示，该酶由两个分子量分别为130,000和55,000的不等亚基组成。已证明纯化后的酶高度不稳定。布氏梨形孢的氨甲酰磷酸合酶以氨而非L-谷氨酰胺作为主要氮供体，并且不需要N-乙酰-L-谷氨酸激活，但它确实需要游离的Mg2+以达到最大活性。动力学研究表明，该酶对氨（Km 6.34 mM）、碳酸氢盐（Km 10.5 mM）和ATP·2Mg2+（Km 0.93 mM）呈双曲线行为。该酶活性的最适pH为7.4 - 7.8。布氏梨形孢氨甲酰磷酸合酶的转变温度为38.5℃。它对鸟氨酸、半胱氨酸、甘氨酸、肌苷酸、二硫苏糖醇、甘油、尿苷一磷酸、尿苷二磷酸和尿苷三磷酸完全不敏感。该酶与5 mM N-乙基马来酰亚胺反应会受到抑制。
