School of Molecular Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia.
Institute of Photonics and Advanced Sensing (IPAS), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.
Biochem J. 2021 Aug 13;478(15):3047-3062. doi: 10.1042/BCJ20210455.
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.
单价阳离子（M+）激活酶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描述其潜在的分子细节。硫解酶是一种普遍存在且高度保守的酶家族，既包含 K+ 激活的成员，也包含 K+ 非依赖的成员。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+ 激活工程化到 K+ 非依赖的硫解酶中。据我们所知，这是首次将 K+ 激活工程化到酶中的演示，表明蛋白质具有可塑造性，可以容纳 M+离子作为别构调节剂。我们表明，该酶类的几个蛋白质结构特征编码了 K+ 激活。具体来说，底物结合位点附近的两个残基足以实现 K+ 激活：一个酪氨酸残基用于完成 K+ 配位球，一个谷氨酸残基为结合的 K+ 离子提供补偿电荷。除此之外，一个远端残基对于定位与底物形成直接氢键的 K+ 配位水分子很重要。带正电荷残基与底物之间的阳离子-π 相互作用的稳定性取决于围绕底物结合位点的环的构象。我们的结果表明，这种阳离子-π 相互作用有效地取代了 K+ 激活，因此在 K+ 激活的硫解酶中被破坏。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。总之，我们的结构、生化和生物信息学工作为 M+ 离子如何能够别构激活酶提供了重要的机制见解。
