The binding of l-serine to phosphoglycerate
dehydrogenase from Escherichia coli displays elements
of both positive and negative cooperativity. At pH 7.5,
∼2 mol of serine are bound per mole of tetrameric enzyme.
A substantial degree of positive cooperativity is seen
for the binding of the second ligand, but the binding of
the third and fourth ligand display substantial negative
cooperativity. The data indicate a state of ∼50% inhibition
when only one serine is bound and ∼80–90% inhibition
when two serines are bound. This is consistent with the
tethered domain hypothesis that has been presented previously.
Comparison of the data derived directly from binding stoichiometry
to the binding constants determined from the best fit to
the Adair equation, produce a close agreement, and reinforce
the general validity of the derived binding constants.
The data also support the conclusion that the positive
cooperativity between the binding to the first and second
site involves binding sites at opposite interfaces over
110 Å apart. Thus, an order of binding can be envisioned
where the binding of the first ligand initiates a conformational
transition that allows the second ligand to bind with much
higher affinity at the opposite interface. This is followed
by the third ligand, which binds with lesser affinity to
one of the two already occupied interfaces, and in so doing,
completes a global conformational transition that produces
maximum inhibition of activity and an even lower affinity
for the fourth ligand, excluding it completely. Thus, maximal
inhibition is accomplished with less than maximal occupancy
of effector sites through a mechanism that displays strong
elements of both positive and negative cooperativity.