Stec B, Abraham R, Giroux E, Kantrowitz E R
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Protein Sci. 1996 Aug;5(8):1541-53. doi: 10.1002/pro.5560050810.
The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.
猪肾果糖-1,6-二磷酸酶（EC 3.1.3.11）的活性位点位于亚基之间，一条链上的精氨酸-243与相邻链活性位点中的果糖-1,6-二磷酸或果糖-2,6-二磷酸相互作用。在本研究中，我们展示了精氨酸-243被丙氨酸取代的该酶突变体版本的X射线结构，其在T态和R态变构状态下均已结晶。改变后的酶的动力学特征表明，镁结合以及AMP的抑制作用略有不同；对底物果糖-1,6-二磷酸的亲和力降低了10倍，对抑制剂果糖-2,6-二磷酸的亲和力降低了1000倍（吉鲁克斯E、威廉姆斯MK、坎特罗维茨ER，1994年，《生物化学杂志》269:31404 - 31409）。X射线结构显示，与野生型酶相比，活性位点的组织没有重大变化，并且这些结构证实了涉及赖氨酸-269和赖氨酸-274的分子动力学模拟的预测。对T态结构的两个独立模型的比较揭示了结合的AMP分子构象的微小但显著的变化以及与抑制剂存在相关的活性位点的小重组。突变酶动力学性质的差异表明精氨酸-243在果糖-1,6-二磷酸酶功能中的关键重要性。使用精氨酸-243→丙氨酸酶的X射线结构进行的计算表明，野生型酶中精氨酸-243的作用本质上主要是静电作用。
