Central Research and Development Department, E.I. du Pont de Nemours and Company, Experimental Station, Wilmington, DE 19898, USA.
EMBO J. 1984 Dec 1;3(12):2737-43. doi: 10.1002/j.1460-2075.1984.tb02204.x.
In vitro mutagenic techniques have generated an asp-->glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C-->A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.
在体外诱变技术中，已经产生了红假单胞菌核酮糖 1,5-二磷酸羧化酶的残基 198 附近的天冬氨酸-谷氨酸取代，该残基紧邻氨基甲酰基-二价金属离子结合位点。编码链上的单个 C->A 核苷酸变化产生了突变体，并在表达质粒 pRR2119 上引入了一个新的 EcoRI 限制位点。尽管羧化酶：加氧酶的比例保持不变，但突变酶的动力学特性略有改变。酶-激活剂氨基甲酰基-Mn-2-羧基阿拉伯糖醇 1,5-二磷酸和酶-激活剂氨基甲酰基-Mn-4-羧基阿拉伯糖醇 1,5-二磷酸的四元复合物的 e.p.r. 光谱对于突变体和野生型酶来说是不同的，这表明金属离子处于略微改变的环境中。这些发现与以下假设一致，除了赖氨酸 201 上的氨基甲酰基外，天冬氨酸 198 的羧基也有助于形成二价金属离子结合位点。
