Baxter Nicola J, Olguin Luis F, Golicnik Marko, Feng Guoqiang, Hounslow Andrea M, Bermel Wolfgang, Blackburn G Michael, Hollfelder Florian, Waltho Jonathan P, Williams Nicholas H
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14732-7. doi: 10.1073/pnas.0604448103. Epub 2006 Sep 21.
Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using (19)F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF(3)(-) mimics the transferring PO(3)(-) moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of beta-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.
确定酶如何在反应途径中稳定高能物种是解释其巨大速率加速的核心。β-磷酸葡萄糖变位酶催化β-葡萄糖-1-磷酸异构化为β-葡萄糖-6-磷酸，并且在稳定高能五配位磷正烷中间体方面具有独特能力，该中间体在酶活性位点中足以被直接观察到。利用¹⁹F-NMR和动力学分析，我们报告所形成的复合物不是假定的高能反应中间体，而是一种看似相似的过渡态类似物，其中MgF₃⁻模拟转移的PO₃⁻部分。在此，我们展示了溶液中与酶活性位点结合的金属离子 - 氟化物复合物的详细表征，揭示了氟化物抑制β-磷酸葡萄糖变位酶的分子机制。这种NMR方法在识别氟化物复合物与蛋白质之间的特定相互作用以及解决X射线晶体学无法区分的结构归属方面具有普遍应用。
