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 βPGMWT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1H, 15N and 13C backbone resonance assignments of the P146A variant (βPGMP146A) in its substrate-free form, where the K145-A146 peptide bond adopts a trans conformation in contrast to all crystal structures of βPGMWT, where the K145-P146 peptide bond is cis. In βPGMP146A 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 βPGMP146A and βPGMWT 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 MgII ion and the L44-L53 loop responsible for substrate discrimination.
Keywords: Backbone resonance assignment; General acid–base catalysis; Phosphoryl transfer enzyme; Transverse relaxation optimised spectroscopy; Triple-labelled protein.