Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, CNRS and Aix-Marseille Université , 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
J Am Chem Soc. 2012 May 23;134(20):8368-71. doi: 10.1021/ja301802r. Epub 2012 May 8.
When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H(2) oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site.
当酶被优化用于生物技术目的时，目标通常是提高稳定性或催化效率。然而，许多酶可逆地转化其底物和产物，如果人们对仅一个方向的催化感兴趣，可能需要防止逆反应。在其他情况下，可逆性可能是有利的，因为只有能够在两个方向上都起作用的酶才能在低热力学驱动力的条件下以高速率进行周转。因此，理解复杂酶中可逆性的基本机制应该有助于对这些蛋白质进行合理的工程设计。在这里，我们专注于 NiFe 氢化酶，它催化 H(2)氧化和生成，并阐明了控制催化偏置（两个方向的最大速率比）的机制。出乎意料的是，我们发现这种偏置主要不是由活性位点的氧化还原性质决定的，而是由远离活性位点的蛋白质部位发生的步骤决定的。我们证明了一种调节氧化还原酶催化偏置的新策略，它包括调节仅在反应的一个方向上受到限制的步骤的速率，而不改变活性位点的性质。
