Hartman F C, Larimer F W, Mural R J, Machanoff R, Soper T S
Biochem Biophys Res Commun. 1987 Jun 30;145(3):1158-63. doi: 10.1016/0006-291x(87)91558-0.
Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.
先前的报道为红螺菌二磷酸核酮糖羧化酶/加氧酶活性位点存在Glu-48提供了间接证据。通过定点诱变将Glu-48替换为谷氨酰胺，直接检验了这种可能性。这种单一氨基酸取代并不妨碍亚基缔合或配体结合。然而，Glu-48突变体的催化活性严重不足，其催化常数仅为野生型酶的0.05%。这些结果表明Glu-48位于活性位点，并表明它发挥着功能作用。结合先前的研究，Glu-48必需性的发现表明活性位点位于亚基之间的界面处。
