The Relationship between Enzyme Conformational Change, Proton Transfer, and Phosphoryl Transfer in β‑Phosphoglucomutase

Molecular details for the timing and role of proton transfer in phosphoryl transfer reactions are poorly understood. Here, we have combined QM models, experimental NMR measurements, and X-ray structures to establish that the transition of an archetypal phosphoryl transfer enzyme, βPGM, from a very closed near-attack conformation to a fully closed transition state analogue (TSA) conformation triggers both partial proton transfer from the general acid–base residue to the leaving group oxygen and partial dissociation of the transferring phosphoryl group from the leaving group oxygen. Proton transfer continues but is not completed throughout the reaction path of the phosphoryl transfer with the enzyme in the TSA conformation. Moreover, using interacting quantum atoms (IQA) and relative energy gradient (REG) analysis approaches, we observed that the change in the position of the proton and the corresponding increased electrostatic repulsion between the proton and the phosphorus atom provide a stimulus for phosphoryl transfer in tandem with a reduction in the negative charge density on the leaving group oxygen atom. The agreement between solution-phase 19F NMR measurements and equivalent QM models of βPGMWT and βPGMD10N TSA complexes confirms the protonation state of G6P in the two variants, validating the employed QM models. Furthermore, QM model predictions of an AlF4 distortion in response to the proton position are confirmed using high resolution X-ray crystal structures, not only providing additional validation to the QM models but also further establishing metal fluorides as highly sensitive experimental predictors of active-site charge density distributions.

人们对磷酰基转移反应中质子转移的时序与分子机制尚缺乏充分认知。本研究结合量子力学（Quantum Mechanics, QM）模型、实验核磁共振（Nuclear Magnetic Resonance, NMR）测量结果与X射线晶体结构，证实经典磷酰基转移酶β磷酸葡萄糖变位酶（β-phosphoglucomutase, βPGM）从高度闭合的近攻击构象转变为完全闭合的过渡态类似物（transition state analogue, TSA）构象时，会同时触发广义酸碱残基向离去基团氧原子发生部分质子转移，以及转移中的磷酰基团与离去基团氧原子发生部分解离。在过渡态类似物构象的酶催化下，磷酰基转移反应路径全程中质子转移仍在持续，但并未完全完成。此外，通过相互作用量子原子（interacting quantum atoms, IQA）与相对能量梯度（relative energy gradient, REG）分析方法，我们发现质子位置的改变及其引发的质子与磷原子间静电排斥增强，与离去基团氧原子负电荷密度的降低协同作用，共同推动磷酰基转移反应。溶液相氟-19核磁共振（19F NMR）测量结果与βPGM野生型（βPGMWT）及βPGM D10N突变体（βPGMD10N）过渡态类似物复合物的等效QM模型之间的一致性，验证了两种变体中葡萄糖-6-磷酸（glucose-6-phosphate, G6P）的质子化状态，同时证实了本研究采用的QM模型的合理性。进一步而言，QM模型对四氟铝根（AlF4⁻）构象随质子位置改变发生畸变的预测，通过高分辨率X射线晶体结构得到了验证。这不仅为QM模型提供了额外的实验支撑，还进一步确立了金属氟化物可作为活性位点电荷密度分布的高灵敏实验探针。