Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity.
Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity.
复制标题
对来自百日咳博德特氏菌的钙调素敏感腺苷酸环化酶的假定 ATP 结合域中的赖氨酸 58 进行定点诱变，从而消除了催化活性。
DOI：
10.1021/bi00433a005
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Storm,DR
中科院分区：
文献类型：
--
作者：
Au,DC;Masure,HR;Storm,DR
道格拉斯C. Au，H. Robert Masure和丹尼尔R. Storm** Department of Pharmacology，SJ-30，School of Medicine，University of华盛顿，西雅图，华盛顿98195 Received January 4，1989摘要：一个2.7-kb的cya A基因片段编码来自百日咳杆菌的钙调素敏感的腺苷酸环化酶的氨基末端，该基因片段被置于lac启动子的控制下，以便在大肠杆菌中表达。异丙基/SD-硫代半乳糖苷诱导后，在E.杆菌表达载体指导合成90-kDa多肽，该多肽被针对B的催化亚基产生的兔多克隆抗体识别。百日咳腺苷酸环化酶。cya A基因产物的推导的氨基酸序列的检查揭示了在许多ATP结合蛋白中发现的ATP结合结构域的一致序列的同源性序列。基于对核苷酸结合蛋白的分析，保守的赖氨酸残基与ATP的结合有关。B中一个假定的ATP结合域。百日咳腺苷酸环化酶在58位具有类似的赖氨酸残基。为了检测B.百日咳腺苷酸环化酶是酶活性的关键残基，它被甲硫氨酸取代。E.将突变体cya A基因转化大肠杆菌细胞，并对表达产物进行了鉴定。突变蛋白既没有表现出基础酶活性，也没有表现出钙调素刺激的酶活性，这表明赖氨酸58在酶催化中起着关键作用。腺苷酸环化酶催化ATP形成3 ′，5 ′-cAMP和无机焦磷酸。由于cAMP是必不可少的各种细胞反应的调制，阐明腺苷酸环化酶的反应机制一直是一个非常感兴趣的主题。已经进行了由细菌和哺乳动物腺苷酸环化酶催化的反应的立体化学研究（Gerlt等人，1980; Eckstein等人，1981年）。在这两种情况下，环化反应发生在α-磷的构型反转。因此，酶反应机理似乎涉及3 '-羟基对α-磷的直接亲核攻击，而不形成腺苷酸化的酶中间体。根据这些结果，Gerlt et al.（1980）提出活性位点的碱性氨基酸参与ATP的3 '-羟基的电离。此外，我们实验室以前的研究已经表明，在哺乳动物腺苷酸环化酶的活性位点存在氨基，其与2 '，3'-二醛ATP形成希夫碱（Westcott et al.，1980年）。百日咳博德特氏菌是百日咳的病原体，其产生的几种毒力因子在百日咳的发病机制中起重要作用（韦斯& Falkow，1986; Wardlaw & Parton，1988）。这些因子之一是在细胞外释放到培养物上清液中的活性腺苷酸环化酶（Hewlett等人，1976; Hewlett & Wolff，1976; Shattuck等人，1985年）。这种酶具有两种不寻常的特性：它被钙调蛋白（CaM）强烈激活，1而钙调蛋白（CaM）不存在于细菌中（Wolff等人，1980），并且它可以侵入动物细胞（Confer & Eaton，1982; Hanski & Farfel，1985; Shattuck & Storm，1985）。编码B的基因。百日咳腺苷酸环化酶已被克隆和测序（Glaser等，1988年a）。这项工作得到了NIH Grant GM 31708的支持。DCA由美国糖尿病博士后奖学金支持; HRM由NIH博士后奖学金NS-07985支持。对谁...
Douglas C. Au, H. Robert Masure, and Daniel R. Storm** Department of Pharmacology, SJ-30, School of Medicine, University of Washington, Seattle, Washington 98195 Received January 4, 1989 abstract: A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placedunder the control of the lac promoterfor expression in Escherichia coli. Following induction with isopropyl/SD-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylatecyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulinstimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis..^^. denylate cyclase catalyzes the formation of 3', 5'-cAMP and inorganic pyrophosphate from ATP. Since cAMP is essential for the modulation of a variety of cellular responses, elucidation of the reaction mechanism for adenylate cyclase has been a subject of great interest. Stereochemical studies of the reactions catalyzed by both the bacterial and the mammalian adenylate cyclasehave been conducted (Gerlt et al., 1980; Eckstein et al., 1981). In both cases, the cyclization reaction occurred with inversion of configuration at the a-phosphorus. The enzyme reaction mechanism therefore appears to involvedirect nucleophilic attack of the 3'-hydroxyl group on the a-phosphorus, with no formation of an adenylated enzyme intermediate. On the basis of these results, Gerlt et al.(1980) proposed that a basic amino acid at the active site participates in the ionization of the 3'-hydroxyl group of ATP. In addition, previous studies from our laboratory have shown that there is an amino group at the active site of the mam-malian adenylate cyclase that forms a Schiff base with 2', 3'-dial ATP (Westcott et al., 1980). Bordetella pertussis, the etiologic agent of whoopingcough, produces several virulence factorsimplicated in the patho-genesis of the disease (Weiss & Falkow, 1986; Wardlaw & Parton, 1988). One of these factors is an active adenylate cyclase that is released extracellularly into the culture su-pernatant (Hewlett et al., 1976; Hewlett & Wolff, 1976; Shattuck et al., 1985). This enzyme exhibits two unusual properties: it is strongly activated by calmodulin (CaM), 1 which is notpresent in bacteria (Wolff et al., 1980), and it can invade animal cells (Confer & Eaton, 1982; Hanski & Farfel, 1985; Shattuck & Storm, 1985). The gene encoding the B. pertussis adenylate cyclase has been cloned and sequenced (Glaser et al., 1988a). Biochemical fThis work was supported by NIH Grant GM 31708. DCA was supported by an American Diabetes postdoctoral fellowship; HRM was supported by NIH Postdoctoral Fellowship NS-07985.* To whom …