Xu Y W, Moréra S, Janin J, Cherfils J
Laboratoire d'Enzymologie et de Biochimie Structurales, Unité Propre de Recherche 9063 Centre National de la Recherche Scientifique, Gif Sur Yvette, France.
Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3579-83. doi: 10.1073/pnas.94.8.3579.
Nucleoside diphosphate kinase reversibly transfers the gamma-phosphate of ATP onto its active site histidine. We have investigated the transition state of histidine phosphorylation with the high-resolution crystal structures of the enzyme from Dictyostelium discoideum with MgADP and either aluminium or beryllium fluoride. The bound aluminium fluoride species is the neutral species AlF3 and not the more common AlF4-. AlF3 forms a trigonal bipyramid that makes it an accurate analog of the transition state of the gamma-phosphate of ATP undergoing transfer to the catalytic histidine. Its axial ligands are a histidine nitrogen and a beta-phosphate oxygen. Beryllium fluoride also binds at the same position and with the same ligands but in a tetrahedral geometry resembling the Michaelis complex rather than the transition state. The two x-ray structures show explicit enzyme-substrate interactions that discriminate between the ground and the transition states of the reaction. They also illustrate the partially dissociative geometry of the transition state of phosphoryl transfer and demonstrate the potential applications of metallofluorides for the study of kinase mechanisms.
核苷二磷酸激酶可将ATP的γ-磷酸基团可逆地转移至其活性位点的组氨酸上。我们利用盘基网柄菌的该酶与MgADP以及氟化铝或氟化铍的高分辨率晶体结构，研究了组氨酸磷酸化的过渡态。结合的氟化铝物种是中性物种AlF3，而非更常见的AlF4-。AlF3形成一个三角双锥，使其成为ATP的γ-磷酸基团向催化性组氨酸转移过程中过渡态的精确类似物。其轴向配体是一个组氨酸氮原子和一个β-磷酸氧原子。氟化铍也结合在相同位置且具有相同配体，但呈四面体几何构型，类似于米氏复合物而非过渡态。这两个X射线结构展示了明确的酶-底物相互作用，这些相互作用区分了反应的基态和过渡态。它们还阐明了磷酰转移过渡态的部分解离几何构型，并证明了金属氟化物在激酶机制研究中的潜在应用。
