Leib T K, Gerlt J A
J Biol Chem. 1983 Nov 10;258(21):12982-7.
Deletions of large portions of the carboxyl-terminal end of the adenylate cyclase (ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1) from Salmonella typhimurium do not significantly affect the enzymatic activity exhibited by the shortened polypeptide. The deletion mutations were generated by nuclease Bal31 digestion from the 3'-end of the cya gene fragment cloned by Wang et al. (Wang, J. Y.-J., Clegg, D. O., and Koshland, D.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4684-4688); the shortened cya genes were inserted in pBR322 and used to transform a cya- strain of Escherichia coli. The original gene fragment encodes for an enzymatically active polypeptide having an apparent molecular weight of 77,000. Mutant polypeptides as small as 46,000 Da were found to retain significant enzymatic activity and to confer several cya+ phenotypes on the E. coli host. More extensive deletions resulting in polypeptides as small as 33,000 Da did not have assayable amounts of adenylate cyclase activity, but the biochemical properties of the transformed cya- host implicate the presence of low levels of enzymatic activity. These data suggest that the structure of the intact enzyme is composed of discrete functional domains. Such a structure for this adenylate cyclase should both facilitate investigations of the chemical mechanism of the reaction and allow structure-function relationships in this physiologically important enzyme to be investigated on a molecular level.
鼠伤寒沙门氏菌腺苷酸环化酶（ATP 焦磷酸裂解酶（环化），EC 4.6.1.1）羧基末端的大部分缺失，对缩短后的多肽所表现出的酶活性没有显著影响。缺失突变是通过核酸酶 Bal31 从 Wang 等人克隆的 cya 基因片段的 3' 末端进行消化产生的（Wang, J. Y.-J., Clegg, D. O., and Koshland, D.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4684-4688）；缩短后的 cya 基因被插入到 pBR322 中，并用于转化大肠杆菌的 cya-菌株。原始基因片段编码一种表观分子量为 77,000 的具有酶活性的多肽。发现小至 46,000 Da 的突变多肽仍保留显著的酶活性，并赋予大肠杆菌宿主几种 cya+表型。更广泛的缺失导致产生小至 33,000 Da 的多肽，其腺苷酸环化酶活性无法检测到，但转化后的 cya-宿主的生化特性表明存在低水平的酶活性。这些数据表明完整酶的结构由离散的功能域组成。这种腺苷酸环化酶的结构既应有助于对反应的化学机制进行研究，又能在分子水平上研究这种生理上重要的酶的结构与功能关系。
