Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2011 Jun 28;108(26):10520-5. doi: 10.1073/pnas.1104989108. Epub 2011 Jun 13.
A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10(13) s(-1) when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.
越来越多的数据支持蛋白质运动在酶催化中的作用。特别是，酶能够采样催化相关的构象亚稳态，这被用来模拟动力学和光谱学数据。然而，快速相互转化的构象与催化化学步骤之间的直接实验联系仍然很少。我们在这里报告了一系列热稳定型醇脱氢酶（ht-ADH）催化的氢化物转移步骤的动力学分析和表征，提供了证据表明，当酶在其最佳温度范围以下运行时，Arrhenius 前因子值大大高于预期值约 10(13) s(-1)。当酶在高温下运行时，ht-ADH 反应恢复正常的 Arrhenius 行为。一个简单的模型表明，降低温度会改变 ht-ADH 变体在催化相关构象空间区域采样的能力，从而可以重现可用数据。这些发现表明，由降低温度和单个活性位点疏水性侧链突变的组合条件产生的 landscapes 受损。更广泛的意义是，最佳的酶功能需要维持相对平滑的 landscape，以最小化低能量陷阱。
