Alonso E, Rubio V
Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas (Centro Asociado del Consejo Superior de Investigaciones Científicas), Amadeo de Saboya, Spain.
Eur J Biochem. 1995 Apr 15;229(2):377-84. doi: 10.1111/j.1432-1033.1995.tb20478.x.
Two ATP molecules are used in the reaction catalyzed by carbamoyl-phosphate synthetase I. One molecule (ATPA) phosphorylates HCO3- and the other (ATPB) phosphorylates carbamate. Carbamoyl-phosphate synthetase I is a 160-kDa polypeptide consisting of a 40-kDa N-terminal moiety and a 120-kDa C-terminal moiety, the latter being composed of two similar halves of molecular mass 60 kDa. We showed [Alonso, E., Cervera, J., García-España, A., Bendala, E. & Rubio, V. (1992) J. Biol. Chem. 267, 4524-4532] that Fe.ATP bound at the site for ATPB catalyzes the oxidative inactivation of carbamoyl-phosphate synthetase I in a model oxidative system consisting of Fe3+, ascorbate, and O2, and we detected ATP-promoted oxidative cleavage of the enzyme. We now provide further evidence indicating that this cleavage is catalyzed by bound Fe.ATPB, and we demonstrate that the enzyme is cleaved at seven points, which we identify as residues 1002, 1064, 1083, 1128, 1200, 1242, and 1270. All these cleavage points are confined within and distributed throughout the more N-terminal 40-kDa region of the C-terminus of the 120-kDa moiety. Thus, this 40-kDa region contains the ATPB site, is folded as a globular domain with the polypeptide recurring several times towards the nucleotide, and appears to be a modular unit equivalent to carbamate kinase, with full responsibility for ATPB binding and carbamate phosphorylation. The present results and our previous demonstration [Rodríguez-Aparicio, L., Guadalajara, A.M. & Rubio, V. (1989) Biochemistry 28, 3070-3074] of the binding of N-acetyl-L-glutamate in the C-terminal 20-kDa region, strongly support the idea that each homologous half of the 120-kDa moiety of carbamoyl-phosphate synthetase I is composed of a 40-kDa ATP-binding domain and a 20-kDa domain that, in the carboxyl half, is the regulatory domain.
在氨甲酰磷酸合成酶I催化的反应中会消耗两个ATP分子。一个分子（ATP A）使HCO3-磷酸化，另一个（ATP B）使氨基甲酸盐磷酸化。氨甲酰磷酸合成酶I是一种160 kDa的多肽，由一个40 kDa的N端部分和一个120 kDa的C端部分组成，后者又由两个分子量为60 kDa的相似部分构成。我们已经证明[阿隆索，E.，塞尔韦拉，J.，加西亚 - 埃斯帕尼亚，A.，本达拉，E. & 鲁维奥，V.（1992年）《生物化学杂志》267，4524 - 4532]，结合在ATP B位点的Fe·ATP在由Fe3 +、抗坏血酸盐和O2组成的模型氧化系统中催化氨甲酰磷酸合成酶I的氧化失活，并且我们检测到了ATP促进的该酶的氧化裂解。我们现在提供进一步的证据表明这种裂解是由结合的Fe·ATP B催化的，并且我们证明该酶在七个位点被裂解，我们将这些位点确定为第1002、1064、1083、1128、1200、1242和1270位氨基酸残基。所有这些裂解位点都局限于120 kDa部分C端更N端的40 kDa区域内并分布于其中。因此，这个40 kDa区域包含ATP B位点，折叠成一个球状结构域，多肽朝着核苷酸方向重复出现几次，并且似乎是一个等同于氨基甲酸盐激酶的模块化单元，完全负责ATP B的结合和氨基甲酸盐的磷酸化。目前的结果以及我们之前[罗德里格斯 - 阿帕里西奥，L.，瓜达拉哈拉，A.M. & 鲁维奥，V.（1989年）《生物化学》28，3070 - 3074]关于N - 乙酰 - L - 谷氨酸在C端20 kDa区域结合的证明，有力地支持了这样一种观点，即氨甲酰磷酸合成酶I的120 kDa部分的每个同源半部分都由一个40 kDa的ATP结合结构域和一个20 kDa的结构域组成，在羧基端的那个半部分是调节结构域。
