Phosphate plays a crucial role in biology because of the stability of the phosphate ester bond. To overcome this inherent stability, enzymes that catalyze phosphoryl transfer reactions achieve enormous rate accelerations to operate on biologically relevant time scales, and the mechanisms that underpin catalysis have been the subject of extensive debate. In an archetypal system, β-phosphoglucomutase catalyzes the reversible isomerization of β-glucose 1-phosphate and glucose 6-phosphate via two phosphoryl transfer steps using a β-glucose 1,6-bisphosphate intermediate and a catalytic MgII ion. In the present work, a variant of β-phosphoglucomutase, where the aspartate residue that acts as a general acid–base is replaced with asparagine, traps highly stable complexes containing the β-glucose 1,6-bisphosphate intermediate in the active site. Crystal structures of these complexes show that, when the enzyme is unable to transfer a proton, the intermediate is arrested in catalysis at an initial stage of phosphoryl transfer. The nucleophilic oxygen and transferring phosphorus atoms are aligned and in van der Waals contact, yet the enzyme is less closed than in transition-state (analogue) complexes, and binding of the catalytic MgII ion is compromised. Together, these observations indicate that optimal closure and optimal MgII binding occur only at higher energy positions on the reaction trajectory, allowing the enzyme to balance efficient catalysis with product dissociation. It is also confirmed that the general acid–base ensures that mutase activity is ∼103 fold greater than phosphatase activity in β-phosphoglucomutase.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)