Yang J K, Epstein W
J Biol Chem. 1983 Mar 25;258(6):3750-8.
Adenylate cyclase of Escherichia coli K12 has been purified 17,000-fold to near homogeneity from a 5-fold overproducing strain. One major band of Mr = 92,000 and several minor bands are seen on sodium dodecyl sulfate-polyacrylamide electrophoresis of the purest fractions. Identification of the enzyme with the 92,000-Da protein is based on the correlation of this band with activity when highly purified enzyme is eluted from ADP-sepharose columns. The native enzyme has a molecular weight of 95,000 determined by gel filtration, showing that the enzyme is active as a monomer. The purest enzyme has a specific activity of 700 nmol min-1 mg-1, indicating a turnover number of about 100 min-1. Our data indicate that there are only about 15 molecules of the enzyme in wild type cells of E. coli. In crude extracts, over 80% of the activity is soluble after centrifugation at 100,000 x g, indicating the enzyme is soluble or, at most, loosely membrane bound. The enzyme is only moderately stable in crude extracts and becomes more unstable as purification proceeds. Activity is stabilized by ATP, or at -20 degrees C as an ammonium sulfate precipitate or in 50% glycerol. The enzyme has an absolute requirement for divalent cations. Maximum activity with Mg2+ is reached at 30 mM. Mn2+ is a good substitute; Co2+ activates well at low concentrations but becomes inhibitory at high concentrations; and Ca2+ is a potent inhibitor in the presence of Mg2+. The isoelectric point of the enzyme is 6.1, and its pH optimum is 8.5. The enzyme is inhibited by its substrate, with a Km of about 1 mM and a Ki of about 1.5 mM, and is noncompetitively inhibited by PPi, ADP, GTP, and a number of other compounds. The data suggest that dissociation of PPi from the first enzyme-product complex is the rate-limiting step in the reaction. Activation of the enzyme, inferred to occur in vivo, could be produced by a postulated regulatory effector which speeds release of PPi from the enzyme-product complex.
已从一个产量高出5倍的菌株中，将大肠杆菌K12的腺苷酸环化酶纯化了17000倍，达到近乎均一的程度。在最纯组分的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，可见一条主要的Mr = 92,000条带以及几条次要条带。当从ADP-琼脂糖柱上洗脱高度纯化的酶时，基于该条带与活性的相关性，将该酶鉴定为92,000 Da的蛋白质。通过凝胶过滤测定，天然酶的分子量为95,000，表明该酶以单体形式具有活性。最纯的酶的比活性为700 nmol min-1 mg-1，表明周转数约为100 min-1。我们的数据表明，在大肠杆菌的野生型细胞中，该酶仅有约15个分子。在粗提物中，100,000 x g离心后，超过80%的活性是可溶的，这表明该酶是可溶的，或者最多是松散地与膜结合。该酶在粗提物中仅具有中等稳定性，并且随着纯化的进行变得更加不稳定。ATP可使活性稳定，或者在-20℃下作为硫酸铵沉淀或在50%甘油中活性稳定。该酶绝对需要二价阳离子。Mg2+在30 mM时达到最大活性。Mn2+是良好的替代物；Co2+在低浓度时能很好地激活，但在高浓度时具有抑制作用；而Ca2+在有Mg2+存在时是一种强效抑制剂。该酶的等电点为6.1，最适pH为8.5。该酶被其底物抑制，Km约为1 mM，Ki约为1.5 mM，并且被PPi、ADP、GTP和许多其他化合物非竞争性抑制。数据表明，PPi从第一个酶-产物复合物中的解离是反应中的限速步骤。推断在体内发生的酶的激活，可能由一种假定的调节效应物产生，该效应物加速PPi从酶-产物复合物中的释放。
