Estelle M, Hanks J, McIntosh L, Somerville C
J Biol Chem. 1985 Aug 15;260(17):9523-6.
Site-specific mutagenesis of a cloned gene for ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was used to examine the functional significance of carbamate activation. Lysine 191, the residue involved in carbamate formation, was replaced with a glutamate in order to mimic the anionic nature of the carbamate. The resulting enzyme was capable of binding the six-carbon transition state analog carboxyarabinitol bisphosphate, but completely lacked catalytic activity. In contrast to the wild-type enzyme, carboxyarabinitol bisphosphate binding was not stabilized by divalent metal and CO2. These observations are consistent with a proposed role for the carbamate in binding the metal required for catalysis.
利用来自红螺菌的1,5-二磷酸核酮糖羧化酶/加氧酶克隆基因的位点特异性诱变来研究氨基甲酸酯激活的功能意义。参与氨基甲酸酯形成的赖氨酸191被谷氨酸取代，以模拟氨基甲酸酯的阴离子性质。所得的酶能够结合六碳过渡态类似物羧基阿拉伯糖醇二磷酸，但完全缺乏催化活性。与野生型酶相比，羧基阿拉伯糖醇二磷酸的结合不受二价金属和二氧化碳的稳定。这些观察结果与氨基甲酸酯在结合催化所需金属中的作用一致。
