Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation–π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation–π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

中文翻译：

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将钾活化工程化为生物合成硫解酶

单价阳离子 (M+) 对酶的激活是生物学中普遍存在的现象。尽管如此，很少有基于结构的研究来描述潜在的分子细节。硫解酶是普遍存在且高度保守的酶家族，包含 K+ 激活和 K+ 非依赖性成员。在天然存在的 K+ 激活硫解酶结构的指导下，我们使用基于结构的方法将 K+ 激活工程化为不依赖 K+ 的硫解酶。据我们所知，这是将 K+ 活化工程化到酶中的首次演示，显示了蛋白质作为变构调节剂容纳 M+ 离子的延展性。我们展示了一些蛋白质结构特征在这类酶中编码 K+-活化。具体来说，底物结合位点附近的两个残基足以激活 K+：需要酪氨酸残基来完成 K+ 配位球，谷氨酸残基为结合的 K+ 离子提供补偿电荷。此外，远端残基对于定位与基底形成直接氢键的 K+ 配位水分子很重要。带正电荷的残基与底物之间阳离子-π相互作用的稳定性取决于底物结合位点周围环的构象。我们的结果表明，这种阳离子-π 相互作用有效地覆盖了 K+ 激活，因此在 K+ 激活的硫解酶中不稳定。这些氨基酸的进化保守性为预测硫解酶中的 K+ 激活提供了有希望的特征序列。一起，我们的结构，