Adipose pyruvate carboxylase: amino acid sequence and domain structure deduced from cDNA sequencing.

The complete amino acid sequence of 3T3-L1 adipocyte pyruvate carboxylase (PC) [pyruvate:carbon-dioxide ligase (ADP-forming), EC 6.4.1.1] has been deduced from sequencing overlapping cDNA clones obtained from an adipocyte cDNA library constructed in the lambda Zap vector. The encoding mRNA for PC promoter contains 4067 nt, including a 3534-nt coding sequence and noncoding regions of 100 and 433 nt at the 5' and 3' ends, respectively. The biotinylated lysine of the encoded PC promoter (1178 amino acids with a calculated M(r) of apocarboxylase = 129,784) is located 35 residues from the COOH-terminal end and, as in most other biotin enzymes, is in the consensus sequence AMKM. The adipocyte PC is closely similar (53% identity) to the yeast enzyme and contains different segments that are homologous with regions from the biotin carboxylase component of Escherichia coli acetyl-CoA carboxylase, the keto acid-binding subunits of Propionibacterium shermanii oxaloacetate transcarboxylase and Klebsiella pneumoniae oxaloacetate decarboxylase, and to the biotin carboxyl-carrier protein of the bacterial biotin enzymes. In addition to the putative mitochondrial targeting signal, functional domains are readily identifiable in the sequence and are in the following order: biotin carboxylase-carboxyltransferase-biotin carboxyl-carrier protein, as proposed for yeast PC.

研究人员从以λ Zap载体（lambda Zap vector）构建的脂肪细胞cDNA文库中分离得到重叠互补DNA（complementary DNA, cDNA）克隆并完成测序，由此推导获得3T3-L1脂肪细胞丙酮酸羧化酶（pyruvate carboxylase, PC）[丙酮酸:二氧化碳连接酶（ADP形成型），EC 6.4.1.1]的完整氨基酸序列。该编码PC的mRNA全长为4067个核苷酸（nt），其中包含3534 nt的编码序列，以及5'端与3'端分别为100 nt和433 nt的非编码区域。所编码的PC蛋白含1178个氨基酸，其脱辅基形式的理论分子量为129784；该蛋白的生物素化赖氨酸位点距羧基末端35个氨基酸残基，且与绝大多数其他生物素酶一致，该位点处于保守序列AMKM中。该脂肪细胞PC与酵母同源酶的序列一致性达53%，其包含多个与不同蛋白区域同源的片段：这些区域分别来自大肠杆菌乙酰辅酶A羧化酶的生物素羧化酶结构域、谢氏丙酸杆菌草酰乙酸转羧化酶的酮酸结合亚基、肺炎克雷伯菌草酰乙酸脱羧酶的对应区域，以及细菌生物素酶的生物素羧基载体蛋白。除推测存在的线粒体靶向信号外，该序列中可清晰识别出功能结构域，其排布顺序为：生物素羧化酶-羧基转移酶-生物素羧基载体蛋白，这与酵母PC的已提出的结构域模型一致。