Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
复制标题
牛心脏低分子量磷酸酪氨酰蛋白磷酸酶的克隆、表达及催化机制。
DOI：
10.1021/bi00121a019
复制
发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
VanEtten,RL
中科院分区：
文献类型：
--
作者：
Wo,YY;Zhou,MM;Stevis,P;Davis,JP;Zhang,ZY;VanEtten,RL
化学系，普渡大学，西拉斐特，印第安纳州47907-1393接收1991年7月22日;修订的Mandarin pt接收1991年11月6日摘要：一组哺乳动物的第一个代表，低分子量磷酸酪氨酰蛋白磷酸酶被克隆，测序并在大肠杆菌中表达。使用基于纯化的酶的氨基酸序列的61-mer寡核苷酸探针，从牛心脏cDNA文库中分离出几个重叠的cDNA克隆。获得了一个全长克隆，包括一个27 bp的5 '端非编码区、一个编码157个氨基酸的开放阅读框和一个3'端非翻译区。克隆全长的鉴定与使用来自牛心脏的poly（A）+ mRNA的mRNA印迹实验中获得的结果一致。将编码序列置于噬菌体T7启动子的下游，并在E.杆菌表达的酶是可溶性的，并且具有催化活性，并且容易分离和纯化。重组蛋白具有预期的Mr为18 000（通过SDS-PAGE估计），并且它显示出与针对牛心脏和人胎盘酶的抗血清的交叉反应性。表达蛋白的N端氨基酸序列表明，甲硫氨酸已被去除，导致与从牛组织中分离的酶的序列相同，除了来自组织的蛋白的N端丙氨酸被乙酰化。从磷酸酶催化的反应中捕获了一种动力学活性的磷酸酶中间体。使用31 P NMR，共价中间体被鉴定为半胱氨酰磷酸。通过与丝氨酸酯酶所用的命名法类比，这些酶可被称为半胱氨酸磷酸酶。
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393 Received July 22, 1991; Revised Manuscript Received November 6, 1991 abstract: The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5'noncoding region, an open reading frame encoding theexpected 157 amino acid protein, and an extensive 3'nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly (A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18 000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from thebovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.