β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a β-glucose 1,6-bisphosphate intermediate. Substrate-free βPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into βPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free βPGM_WT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report ¹H, ¹⁵N and ¹³C backbone resonance assignments of the P146A variant (βPGM_P146A) in its substrate-free form, where the K145–A146 peptide bond adopts a trans conformation in contrast to all crystal structures of βPGM_WT, where the K145–P146 peptide bond is cis. In βPGM_P146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect βPGM crystal structures, and a chemical shift comparison between substrate-free βPGM_P146A and βPGM_WT confirms that the solution conformations are very similar, except for the D137–A147 loop. Hence, the isomerisation state of the 145–146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12–T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114–S116), and the D137–A147 loop (V141–A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic Mg^II ion and the L44–L53 loop responsible for substrate discrimination.

中文翻译：

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无底物形式的乳酸乳球菌β-磷酸葡萄糖变位酶的P146A变体的1H，15N和13C骨架共振分配。

β-磷酸葡萄糖突变酶（βPGM）是一种依赖镁的磷酸基转移酶，它通过两个磷酸基转移步骤和一个β-葡萄糖1,6-双磷酸酯中间体催化β-葡萄糖1-磷酸和6-葡萄糖葡萄糖的可逆异构化。不含底物的βPGM是催化循环的重要组成部分，对其动力学的理解将对βPGM的功能性以及酶催化的磷酰基转移产生重要的见解。此前，周围无基材的βPGM的活性位点30个残基_WT被确定为发生广泛毫秒动力学并且是无法分配的。这里，我们报告^1个H，¹⁵ N和^13次C中的P146A变体的主链共振分配（βPGM _P146A）在其无基材的形式，其中，K145-A146肽键采用反式相反构象βPGM的所有晶体结构_WT，其中K145-P146肽键是顺式的。在βPGM中，除17个残基外，_P146A的毫秒级动力学都受到抑制，可以分配92％的骨架共振。使用TALOS-N二级结构预测反映βPGM晶体结构，和不含基底的βPGM之间的化学位移的比较_P146A和βPGM _WT该溶液构象非常相似确认，除了D137-A147回路。因此，145–146肽键的异构化状态对结构的影响很小，但顺式构象在铰链（V12–T16），亲核试剂（D8）和残基协调传递的磷酸基团（D8和S114–S116）和D137–A147环（V141–A142和K145）中触发毫秒级动力学。除了涉及协调催化Mg ^II离子和负责底物识别的L44-L53环的残基的动力学之外，还会发生这些毫秒级的动力学。