Guy H I, Evans D R
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 1994 Sep 23;269(38):23808-16.
The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.
多功能蛋白CAD催化哺乳动物细胞中从头嘧啶生物合成的前三个步骤。谷氨酰胺依赖性氨甲酰磷酸合成酶（CPSase）、天冬氨酸转氨甲酰酶和二氢乳清酸酶活性由一条243 kDa的多肽链承担，该多肽链被组织成由结构域间连接子相连的离散功能结构域。连接链段之一，即连接二氢乳清酸酶和天冬氨酸转氨甲酰酶结构域的DA连接子，异常长（109个残基），并且在所有真核生物物种中长度保守。构建了编码整个243 kDa多肽的质粒（pCK-CAD10）并在大肠杆菌中表达。重组蛋白通过离子交换和凝胶过滤色谱法纯化至同质。纯化后的蛋白具有与天然CAD相近的动力学参数。此外，CPSase活性受到别构调节。凝胶过滤显示重组蛋白与天然CAD具有相同的分子量。因此，这种复杂的哺乳动物蛋白在细菌细胞中表达并正确折叠，尽管其对蛋白酶极度敏感，但仍可完整分离。然后构建了一个缺失DA连接子的缺失突变体。突变蛋白的动力学参数在很大程度上未改变，表明DA连接子对于各个结构域的正确折叠或最佳功能并非必不可少。然而，CPSase结构域热稳定性的显著降低表明连接子有助于稳定该复合物。此外，通过测量外源添加的中间体能够稀释内源性氨甲酰磷酸池的程度来确定的氨甲酰磷酸的通道化，在去除DA连接子后明显降低。因此，尽管各个结构域自主发挥功能，但某些连接子对于CAD中的结构域间相互作用很重要。
