α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction
α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction
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DOI：
10.1073/pnas.1402850111
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发表时间：
2014-08-26
影响因子：
11.1
通讯作者：
Waltho, Jonathan P.
中科院分区：
文献类型：
--
作者：
Jin, Yi;Bhattasali, Debabrata;Waltho, Jonathan P.
β -磷酸葡萄糖糖化酶（β - PGM）利用β - d -葡萄糖1,6-二磷酸（β - G16BP）中间体通过顺序磷酸化转移步骤催化β - d -葡萄糖1-磷酸（β - G1P）异构化为d -葡萄糖6-磷酸（G6P）。合成的β - G1P的氟甲基-内膦酸盐和亚膦酸盐类似物为β - PGM提供了新的1阶过渡态类似物（TSA）配合物，其中含有磷酸基的三氟镁酸盐和四氟铝酸盐代物。在不变的蛋白质构象中，β - G1P - TSA复合物（步骤1）中的β - d -葡萄糖吡喃糖环相对于G6P - TSA复合物（步骤2）翻转并移位。它的赤道羟基与酶直接成氢键，而不是像步骤2那样通过水分子间接成键。在步骤1和步骤2之间，在惰性磷酸盐位点结合磷酸盐的(C) O-P键取向相差约30度。相比之下，催化位点上磷基的轴向O-Mg-O排列的取向仅相差5度，并且代表两个过渡态（TSs）的五个磷键氧的原子实际上是重叠的。步骤1中β G16BP的构象不适合步骤2中相同的不变活性位点，通过磷酸的简单位置交换：TS对齐是通过己糖的构象变化而不是蛋白质的构象变化实现的。
beta-Phosphoglucomutase (beta PGM) catalyzes isomerization of beta-D-glucose 1-phosphate (beta G1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a beta-D-glucose 1,6-bisphosphate (beta G16BP) intermediate. Synthetic fluoromethyle-nephosphonate and methylenephosphonate analogs of beta G1P deliver novel step 1 transition state analog (TSA) complexes for beta PGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the beta-D-glucopyranose ring in the beta G1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C) O-P bond orientation for binding the phosphate in the inert phosphate site differs by similar to 30 degrees between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only similar to 5 degrees, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of beta G16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.