Taylor T C, Andersson I
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
J Mol Biol. 1997 Jan 31;265(4):432-44. doi: 10.1006/jmbi.1996.0738.
The three-dimensional structure of the complex of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco; EC 4.1.1.39) from spinach with its natural substrate ribulose 1,5-bisphosphate (RuBP) has been determined both under activating and non-activating conditions by X-ray crystallography to a resolution of 2.1 A and 2.4 A, respectively. Under activating conditions, the use of calcium instead of magnesium as the activator metal ion enabled us to trap the substrate in a stable complex for crystallographic analysis. Comparison of the structure of the activated and the non-activated RuBP complexes shows a tighter binding for the substrate in the non-activated form of the enzyme, in line with previous solution studies. In the non-activated complex, the substrate triggers isolation of the active site by inducing movements of flexible loop regions of the catalytic subunits. In contrast, in the activated complex the active site remains partly open, probably awaiting the binding of the gaseous substrate. By inspection of the structures and by comparison with other complexes of the enzyme we were able to identify a network of hydrogen bonds that stabilise a closed active site structure during crucial steps in the reaction. The present structure underlines the central role of the carbamylated lysine 201 in both activation and catalysis, and completes available structural information for our proposal on the mechanism of the enzyme.
通过X射线晶体学分别在激活和非激活条件下测定了菠菜中1,5 - 二磷酸核酮糖羧化酶/加氧酶（rubisco；EC 4.1.1.39）与其天然底物1,5 - 二磷酸核酮糖（RuBP）复合物的三维结构，分辨率分别为2.1 Å和2.4 Å。在激活条件下，使用钙而非镁作为激活金属离子使我们能够捕获处于稳定复合物状态的底物用于晶体学分析。激活态和非激活态RuBP复合物结构的比较表明，在酶的非激活形式中底物结合更紧密，这与之前的溶液研究一致。在非激活复合物中，底物通过诱导催化亚基的柔性环区域移动来触发活性位点的隔离。相反，在激活复合物中活性位点部分保持开放，可能在等待气态底物的结合。通过检查结构并与该酶的其他复合物进行比较，我们能够识别出一个氢键网络，该网络在反应的关键步骤中稳定封闭的活性位点结构。目前的结构强调了氨甲酰化赖氨酸201在激活和催化过程中的核心作用，并完善了我们关于该酶作用机制提议的现有结构信息。
