Robertson Angus J, Cruz-Navarrete F Aaron, Wood Henry P, Vekaria Nikita, Hounslow Andrea M, Bisson Claudine, Cliff Matthew J, Baxter Nicola J, Waltho Jonathan P
School of Biosciences, The University of Sheffield, Sheffield, S10 2TN, United Kingdom.
Manchester Institute of Biotechnology and Department of Chemistry, The University of Manchester, Manchester, M1 7DN, United Kingdom.
ACS Catal. 2022 Mar 4;12(5):3149-3164. doi: 10.1021/acscatal.1c05524. Epub 2022 Feb 22.
Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use β-phosphoglucomutase (βPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. βPGM catalyzes the isomerization of β-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. βPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported β-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.
了解支撑酶巨大催化效率的因素是催化和酶设计的基础。酶部分地能够通过利用来自底物非反应部分的结合能来实现高催化效率。特别是，有人提出，底物含有在远端位点配位的非反应性磷酸二阴离子基团的酶，主要利用这种结合能来促进从开放的无活性形式到封闭的活性形式的构象变化，而不是诱导基态去稳定化或稳定过渡态。然而，该模型的详细结构证据有限。在这里，我们使用β-磷酸葡萄糖变位酶（βPGM）来研究在远端位点结合磷酸二阴离子基团、采用封闭的酶形式和催化效率之间的关系。βPGM通过近端位点的磷酸转移反应催化β-葡萄糖1-磷酸异构化为葡萄糖6-磷酸，同时在远端位点配位底物的磷酸二阴离子基团。βPGM具有所测量的最大催化效率之一，并且在其催化循环中经历显著的结构域闭合。我们发现，远端位点的侧链取代导致底物结合减少，从而使封闭的活性形式不稳定，但不足以阻止采用完全封闭的、接近过渡态的构象。此外，我们发现，即使近端位点没有转移的磷酸基团，远端位点磷酸二阴离子基团的结合也会刺激结构域闭合，这解释了先前报道的β-葡萄糖1-磷酸抑制作用。最后，我们的结果支持一种趋势，即涉及磷酸化底物的具有高催化效率的酶对稳定封闭的活性形式有更大的需求。
