Resolving conformational changes that mediate a two-step catalytic mechanism in a model enzyme.
Resolving conformational changes that mediate a two-step catalytic mechanism in a model enzyme.
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解决模型酶中介导两步催化机制的构象变化。
DOI：
10.1101/2023.06.02.543507
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Hekstra,DoekeR
中科院分区：
文献类型：
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作者：
Greisman,JackB;Dalton,KevinM;Brookner,DennisE;Klureza,MargaretA;Sheehan,CandiceJ;Kim,In-Sik;Henning,RobertW;Russi,Silvia;Hekstra,DoekeR
酶通过精确定位底物、辅因子和氨基酸来调节过渡态的自由能，催化生化反应。然而，由于缺乏实验途径，构象动力学的作用仍然知之甚少。这一缺点在大肠杆菌二氢叶酸还原酶（DHFR）中很明显，DHFR是蛋白质动力学催化作用的模型系统，但目前尚不清楚该酶如何调节促进质子和氢化物转移所需的不同活性位点环境。在这里，我们在x射线衍射实验中提出了基于配体、温度和电场的扰动，从而能够识别DHFR中耦合构象的变化。我们确定了一个全局铰链运动和结构重排的局部网络，这些网络由底物质子化参与，以调节溶剂的进入和促进有效的催化。结果表明，DHFR的两步催化机制是由响应底物状态的动态自由能景观引导的。
Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to lack of experimental access. This shortcoming is evident with E. coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we present ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments that enable identification of coupled conformational changes in DHFR. We identify a global hinge motion and local networks of structural rearrangements that are engaged by substrate protonation to regulate solvent access and promote efficient catalysis. The resulting mechanism shows that DHFR’s two-step catalytic mechanism is guided by a dynamic free energy landscape responsive to the state of the substrate.