Simmer J P, Kelly R E, Scully J L, Grayson D R, Rinker A G, Bergh S T, Evans D R
Department of Biochemistry, Wayne State University, Detroit, MI 48201.
Proc Natl Acad Sci U S A. 1989 Jun;86(12):4382-6. doi: 10.1073/pnas.86.12.4382.
Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.
哺乳动物天冬氨酸转氨甲酰酶（天冬氨酸转氨甲酰酶；氨甲酰磷酸：L-天冬氨酸氨甲酰转移酶，EC 2.1.3.2）是一种名为CAD的240 kDa多功能多肽的一部分，该多肽还具有氨甲酰磷酸合成酶和二氢乳清酸酶活性。我们对叙利亚仓鼠CAD cDNA的选定限制性片段进行了测序，这些片段与三种原核天冬氨酸转氨甲酰酶明显同源。这些研究与先前的序列数据相结合，表明CAD的天冬氨酸转氨甲酰酶结构域由924个碱基对编码，质量为34323 Da，pI为9.8。虽然细菌嘧啶生物合成酶是单独的蛋白质，但在哺乳动物中，天冬氨酸转氨甲酰酶结构域通过一个133个氨基酸（14 kDa）的连接子与CAD嵌合体的羧基末端融合，该连接子具有不寻常的氨基酸组成、pI为10.2且具有明显的亲水性。从蛋白水解消化物中分离出的完全活性结构域通过部分氨基酸测序和氨基酸分析进行了表征。胰蛋白酶切割产生了具有20个残基氨基末端延伸的天冬氨酸转氨甲酰酶结构域。流体动力学研究表明，分离出的结构域是一个110 kDa的三聚体，斯托克斯半径为41 Å。哺乳动物天冬氨酸转氨甲酰酶结构域和原核酶具有几乎相同的活性位点残基，并且可能具有相同的三级结构。
