Xi X G, Van Vliet F, Ladjimi M M, De Wannemaeker B, De Staercke C, Piérard A, Glansdorff N, Hervé G, Cunin R
Laboratoire d'Enzymologie, C.N.R.S., Gif-sur-Yvette, France.
J Mol Biol. 1990 Nov 20;216(2):375-84. doi: 10.1016/s0022-2836(05)80328-6.
In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.
在天冬氨酸转氨甲酰酶（ATCase）中，每个调节链与两个催化链相互作用，每个催化链属于不同的三聚体催化亚基（如图1所定义的R1-C1和R1-C4类型的相互作用）。为了研究参与催化位点之间协同相互作用的链间接触，通过定点诱变制备了一系列该酶的修饰形式。氨基酸替换是根据先前描述的一种改变形式的ATCase（pAR5-ATCase）的特性设计的，该酶缺乏催化位点之间的同促协同相互作用。获得的结果（酶动力学、双底物类似物影响和pH研究）表明，R1-C4相互作用对于建立对天冬氨酸具有低亲和力的酶构象（T态）至关重要，因此对于催化位点之间协同性的存在也至关重要。这种相互作用涉及催化链天冬氨酸结合结构域的236-250区域（240s环）和调节链的143-149区域，该区域包含螺旋H3'。
