Kvalnes-Krick K L, Traut T W
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599-7260.
J Biol Chem. 1993 Mar 15;268(8):5686-93.
A cDNA for beta-alanine synthase from rat liver has been isolated, sequenced, and characterized. beta-Alanine synthase clones were isolated from rat liver cDNA libraries in lambda gt11, using affinity-purified polyclonal antibodies against beta-alanine synthase protein. beta-Alanine synthase protein was not expressed with equal efficiency by all clones. One of the expressed fusion proteins has normal specific enzyme activity, and a second has reduced specific activity. Both clones were completely sequenced and yielded identical DNA sequence, except that one clone contained an additional 36 bases of 5' sequence. The various clones of this cDNA code for an EcoRI insert of 1.5 +/- 0.1 kb, and the open reading frame corresponds to a protein of 393 amino acids (M(r) = 44,042), in good agreement with the M(r) of approximately 42,000 for the native enzyme on SDS-gel electrophoresis. An 11-amino acid sequence was obtained from a tryptic peptide of native beta-alanine synthase; 11 codons for these same amino acids were found at the expected site in the sequenced cDNA, and confirm the open reading frame of the beta-alanine synthase cDNA. Chemical analysis of the native enzyme shows 2 zinc atoms per subunit, and the sequence of beta-alanine synthase contains 2 putative zinc-binding site motifs. Comparison of amino acid sequence, deduced from the cDNA sequence, to sequences in the protein data base showed that it is a unique sequence and that it has about 20% identity to aspartate carbamoyltransferase, ornithine carbamoyltransferase, urease, and leucine aminopeptidase; enzymes that bind comparable ligands or have a similar mechanism.
已从大鼠肝脏中分离、测序并鉴定了β-丙氨酸合酶的cDNA。使用针对β-丙氨酸合酶蛋白的亲和纯化多克隆抗体，从λgt11大鼠肝脏cDNA文库中分离出β-丙氨酸合酶克隆。并非所有克隆都能以相同效率表达β-丙氨酸合酶蛋白。其中一个表达的融合蛋白具有正常的比酶活性，另一个的比活性降低。对这两个克隆都进行了完整测序，得到的DNA序列相同，只是其中一个克隆在5'端序列多了36个碱基。该cDNA的各种克隆编码一个1.5±0.1kb的EcoRI插入片段，开放阅读框对应一个393个氨基酸的蛋白质（M(r)=44,042），这与SDS凝胶电泳上天然酶约42,000的M(r)非常一致。从天然β-丙氨酸合酶的胰蛋白酶肽段获得了一个11个氨基酸的序列；在测序的cDNA中预期位置发现了这些相同氨基酸的11个密码子，证实了β-丙氨酸合酶cDNA的开放阅读框。对天然酶的化学分析表明每个亚基含有2个锌原子，β-丙氨酸合酶的序列包含2个推定的锌结合位点基序。将从cDNA序列推导的氨基酸序列与蛋白质数据库中的序列进行比较，结果表明它是一个独特的序列，与天冬氨酸氨甲酰基转移酶、鸟氨酸氨甲酰基转移酶、脲酶和亮氨酸氨肽酶有大约20%的同源性；这些酶结合类似的配体或具有相似的机制。
