The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.
The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.
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DOI：
10.1021/jacs.7b00236
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发表时间：
2017-04-05
影响因子：
15
通讯作者：
Brüschweiler R
中科院分区：
文献类型：
--
作者：
Peng Y;Hansen AL;Bruschweiler-Li L;Davulcu O;Skalicky JJ;Chapman MS;Brüschweiler R
精氨酸激酶（AK）是磷酸原激酶家族的一员，是研究酶循环中所有主要状态的生物分子酶催化的结构和动力学决定因素的模型系统。这些状态是apo状态（无底物）、米氏络合物类似物AK：Arg：Mg·AMPPNP（MCA）、产物络合物类似物AK：pAIE：Mg·ADP（PCA）和过渡态类似物AK：Arg：Mg·ADP：NO3−（TSA）。这些国家的构象动力学研究了由NMR弛豫色散测量的甲基的Ile，Leu，和瓦尔残基在两个静态磁场。尽管所有的状态都经历了大量的μs-ms时间尺度动力学，但只有MCA样品具有类似于TSA的主导激发态，正如这些状态的弛豫色散衍生的化学位移差Δω和平衡化学位移差Δδ之间的强相关性所证明的那样。MCA的平均寿命为36 ms，与TSA样形式的自由能差为8.5 kJ/mol。它示出的米氏络合物类似物的构象能量景观的方式，在室温下，它的通道通过过渡态，从而确定精氨酸的磷酸化反应的限速步骤的形状。相反，TSA的弛豫色散实验表明，它采样的米氏络合物类似物或载脂蛋白状态的结构作为其主导的激发态。这种相互作用的行为表明，TSA的自由能，与所有的配体结合，是低的只有约8.9 kJ/mol比米氏或apo复杂的构象与TSA配体的存在。
Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3− (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of μs–ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.
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影响因子：
0.9
作者：
Davulcu O;Niu X;Brüschweiler-Li L;Brüschweiler R;Skalicky JJ;Chapman MS
通讯作者：
Chapman MS
影响因子：
7.7
作者：
Markley JL;Brüschweiler R;Edison AS;Eghbalnia HR;Powers R;Raftery D;Wishart DS
通讯作者：
Wishart DS
影响因子：
5.6
作者：
Brath, Ulrika;Akke, Mikael
通讯作者：
Akke, Mikael
影响因子：
3.9
作者：
BLETHEN, SL
通讯作者：
BLETHEN, SL
影响因子：
56.9
作者：
Boehr, David D.;McElheny, Dan;Wright, Peter E.
通讯作者：
Wright, Peter E.