Bar-Even Arren, Milo Ron, Noor Elad, Tawfik Dan S
†Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
∥Institute of Molecular Systems Biology, ETH Zurich, Auguste-Piccard-Hof 1, CH-8093 Zurich, Switzerland.
Biochemistry. 2015 Aug 18;54(32):4969-77. doi: 10.1021/acs.biochem.5b00621. Epub 2015 Aug 6.
The pioneering model of Henri, Michaelis, and Menten was based on the fast equilibrium assumption: the substrate binds its enzyme reversibly, and substrate dissociation is much faster than product formation. Here, we examine this assumption from a somewhat different point of view, asking what fraction of enzyme-substrate complexes are futile, i.e., result in dissociation rather than product formation. In Knowles' notion of a "perfect" enzyme, all encounters of the enzyme with its substrate result in conversion to product. Thus, the perfect enzyme's catalytic efficiency, kcat/KM, is constrained by only the diffusion on-rate, and the fraction of futile encounters (defined as φ) approaches zero. The available data on >1000 different enzymes suggest that for ≥90% of enzymes φ > 0.99 and for the "average enzyme" φ ≥ 0.9999; namely, <1 of 10(4) encounters is productive. Thus, the "fast equilibrium" assumption holds for the vast majority of enzymes. We discuss possible molecular origins for the dominance of futile encounters, including the coexistence of multiple sub-states of an enzyme's active site (enzyme floppiness) and/or its substrate. Floppiness relates to the inherent flexibility of proteins, but also to conflicting demands, or trade-offs, between rate acceleration (the rate-determining chemical step) and catalytic turnover, or between turnover rate and accuracy. The study of futile encounters and active-site floppiness may contribute to a better understanding of enzyme catalysis, enzyme evolution, and improved enzyme design.
亨利、米凯利斯和门滕提出的开创性模型基于快速平衡假设：底物与酶可逆结合，且底物解离比产物形成快得多。在此，我们从一个略有不同的角度审视这一假设，即询问有多少比例的酶 - 底物复合物是无效的，也就是会导致解离而非产物形成。在诺尔斯提出的“完美”酶的概念中，酶与底物的所有相遇都会转化为产物。因此，完美酶的催化效率kcat/KM仅受扩散结合速率的限制，无效相遇的比例（定义为φ）趋近于零。关于1000多种不同酶的现有数据表明，对于≥90%的酶，φ>0.99，对于“平均酶”，φ≥0.9999；也就是说，10⁴次相遇中不到1次是有成效的。因此，“快速平衡”假设适用于绝大多数酶。我们讨论了无效相遇占主导的可能分子起源，包括酶活性位点（酶的柔性）及其底物的多种亚状态的共存。柔性不仅与蛋白质的固有灵活性有关，还与速率加速（限速化学步骤）和催化周转之间，或周转速率与准确性之间相互冲突的需求或权衡有关。对无效相遇和活性位点柔性的研究可能有助于更好地理解酶催化、酶进化和改进酶的设计。
