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.