Evans D R, Balon M A
Department of Biochemistry, Wayne State University School of Medicine, Detroit, MI 48201.
Biochim Biophys Acta. 1988 Mar 23;953(2):185-96. doi: 10.1016/0167-4838(88)90023-4.
Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl2, NH4Cl, and the activator N-acetyl-L-glutamate. These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species and a 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH4Cl and N-acetyl-L-glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.
从仓鼠肝线粒体中分离出的氨依赖性氨甲酰磷酸合成酶I（二氧化碳：氨连接酶（形成ADP，氨基甲酰磷酸化），EC 6.3.4.16；原EC 2.7.2.5）由相同的160 kDa多肽链组成。弹性蛋白酶控制的蛋白水解作用依次将该分子切割成少量特定片段。第一次切割导致酶活性完全丧失，并形成一个145 kDa的片段，该片段随后又降解为83 kDa和62 kDa的片段。当在饱和ATP、MgCl2、NH4Cl和激活剂N-乙酰-L-谷氨酸存在的情况下进行蛋白水解时，得到了非常不同的结果。这些配体使该分子对弹性蛋白酶消化的稳定性提高了8倍。此外，仅产生少量的145 kDa片段。相反，该分子最初被切割成一个完全有活性的120 kDa片段和一个40 kDa的蛋白水解片段。在有和没有配体的情况下进行的极限消化中发现了相同的片段，这表明只是弹性蛋白酶的切割顺序不同。对单独和组合存在每种配体时进行的消化的比较表明，虽然NH4Cl和N-乙酰-L-谷氨酸对于分子的最大稳定性是必需的，但消化模式的改变是由MgATP特异性产生的。MgATP诱导的消化模式变化与稳态ATP饱和曲线密切相关，这表明120 kDa片段的产生是由于ATP与活性位点结合所致。MgATP对氨甲酰磷酸合成酶I蛋白水解的影响不是寡聚体结构改变的结果，而是对两个弹性蛋白酶切割位点的保护。根据最近在其他地方确定的一级结构对结果进行了解释。
