Optimal alignment for enzymatic proton transfer:: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution
Optimal alignment for enzymatic proton transfer:: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution
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DOI：
10.1073/pnas.0233793100
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发表时间：
2003-01-07
影响因子：
11.1
通讯作者：
Tong, L
中科院分区：
文献类型：
--
作者：
Jogl, G;Rozovsky, S;Tong, L
在酶催化中，精确定位的功能性是必要条件，米氏复合物的原子坐标可以提供对底物活化的有力见解。在这里，我们专注于磷酸丙糖异构酶催化的异构化反应的初始质子转移，并在近原子分辨率下呈现其米氏络合物与底物磷酸二羟丙酮的晶体结构。活性位点是高度紧凑的，具有催化Glu-165和His-95残基的异常短和分叉的氢键。催化碱Glu-165的羧酸氧与底物的酮和α-羟基碳（C. O约3.0埃），这对于涉及这些中心的质子转移是最佳的。使底物极化的亲电体His-95与底物的O 1和O2（N... O分别小于或等于3.0和2.6埃）。在米氏络合物中，底物在构象上是松弛的：磷酸基团在酮基团的平面之外，并且羟基和酮氧原子不处于顺式构型。亲电赖氨酸-12的E铵基团在底物的酮氧、桥氧和末端磷酸的氧的氢键距离内，这表明该残基在催化和控制活性位点环的灵活性中的作用。
In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the alpha-hydroxy carbons of the substrate (C...O approximate to 3.0 Angstrom), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N...O less than or equal to 3.0 and 2.6 Angstrom, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The E ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.