Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis
Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis
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DOI：
10.1021/bi002499f
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发表时间：
2001-01-30
期刊：
影响因子：
2.9
通讯作者：
Almo, SC
中科院分区：
文献类型：
--
作者：
Fedorov, A;Shi, W;Almo, SC
免疫西林-H[ImmH；(1S)-1-(9-deazahypoxanthin-9-yl)-1，4-dideoxy-1，4-imino-D-ribitol]是一种23 PM的牛嘌呤核苷磷酸化酶抑制剂，专为过渡态模拟物而设计[Miles，R.W.，Tyler，P.C.，Furneaux，R.H.，Bagdassarian，C.K.，and Schramm，V.L.(1998年)BioChemical 37,8615-8621]。使用PNP的晶体和抑制剂通过PNP的反应坐标提供每一步的结构信息。在0.9埃分辨率下对游离的ImMH的X射线晶体结构进行了解析，得到了PNP的络合物。嗯，嗯。PO4在1.5埃分辨率下求解。将这些结构与以前报道的PNP与催化中心的底物和产物类似物的络合物以及实验确定的过渡态结构进行了比较。结合后，ImmH被扭曲为有利于核糖基氧碳正离子形成的构象。核糖基氧碳正离子的核糖失稳和过渡态稳定是通过邻基与磷酸阴离子和核糖基的5‘-羟基相互作用而发生的。次黄嘌呤的离开基团激活涉及与嘌呤环的O6、N1和N7的氢键。有序的水分子提供了通往O6和N7的质子转移桥，并允许这些氢键的可逆形成。在PNP的过渡态模拟络合物中，PNP与催化中心配体之间的接触较短。嗯，嗯。PO4，而不是PNP的米氏络合物。肌苷。SO4或PNP。次黄嘌呤。核糖1-PO4。反应配位运动主要由核糖的碳1‘在相对固定的磷酸基团和嘌呤基团之间的平移控制。嘌呤和嘧啶磷酸核糖转移酶和核苷N-核糖水解酶的作用机制似乎相似。
Immuncillin-H [ImmH; (1S)-1 -(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 Angstrom resolution, and a complex of PNP . ImmH . PO4 was solved at 1.5 Angstrom resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP . ImmH . PO4 than in the Michaelis complexes of PNP . inosine . SO4 or PNP . hypoxanthine . ribose 1-PO4. Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.