Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-13C]-Glycolaldehyde in D2O
Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-13C]-Glycolaldehyde in D2O
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DOI：
10.1021/bi900636c
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发表时间：
2009-06-23
期刊：
影响因子：
2.9
通讯作者：
Richard, John P.
中科院分区：
文献类型：
--
作者：
Go, Maybelle K.;Amyes, Tina L.;Richard, John P.
用H-1核磁共振谱法测定了三磷酸异构酶（TIM）在D2O和pD 7.0下催化羰基碳（[1-C-13]-GA）上以碳-13标记的乙醇醛在亚磷酸根离子存在和不存在时的反应产物分布。我们观察到三种相对快速的磷酸盐激活反应产物（Amyes， T. L., and Richard， J. P.（2007）《生物化学》46,5841-5854）：[2-C-13]-GA由氢分子内转移产生（12%的产物），[2- c -13,2- h -2]-GA由D2O在C-2处加入氘产生（64%的产物），和[1- c -13,2- h -2]-GA由D2O在C-2处加入氘产生（23%的产物）。在没有亚硝酸盐的情况下，慢得多的非活化反应产生了同样的三种产物以及双氘化产物[1- c -13,2,2- h -2(2)]-GA。两种异构化产物（[2- c -13]-GA和[2- c -13,2- h -2]-GA）在未激活和亚磷酸盐激活的反应中以相同的相对产率形成。然而，在未激活的tim催化反应中形成的额外的[1- c -13,2- h -2]-GA和双氘化的[1- c -13,2,2- h -2]-GA被认为是由蛋白质表面的非特异性反应产生的。结果表明，亚磷酸钠影响tim催化[1-C-13]-GA在酶活性位点的反应速率，而不影响产物分布。这与两种反应都发生在不稳定的闭环形式的TIM上的结论是一致的，亚磷酸酯阴离子激活异构化反应是由于利用亚磷酸酯阴离子的固有结合能来稳定活性的闭环酶。
Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-C-13]-GA) catalyzed by triosephosphate isomerase (TIM) in D2O at pD 7.0 in the presence of phosphite dianion and in its absence were determined by H-1 NMR spectroscopy. We observe three products for the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841-5854): [2-C-13]-GA from isomerization with intramolecular transfer of hydrogen (12% of products), [2-C-13,2-H-2]-GA from isomerization with incorporation of deuterium from D2O at C-2 (64% of products), and [1-C-13,2-H-2]-GA from incorporation of deuterium from D2O at C-2 (23% of products). The much slower unactivated reaction in the absence of phosphite results in formation of the same three products along with the doubly deuterated product [1-C-13,2,2-H-2(2)]-GA. The two isomerization products ([2-C-13]-GA and [2-C-13,2-H-2]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activated reactions: However, the additional [1-C-13,2-H-2]-GA and the doubly deuterated [1-C-13,2,2-H-2(2)]-GA formed in the unactivated TIM-catalyzed reaction are proposed to result from nonspecific reaction(s) at the protein surface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution, of the TIM-catalyzed reaction of [1-C-13]-GA at the enzyme active site. They are consistent with the conclusion that both reactions occur at an unstable loop-closed form of TIM and that activation of the isomerization reaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianion to stabilize the active loop-closed enzyme.