Price To Be Paid for Two-Metal Catalysis: Magnesium Ions That Accelerate Chemistry Unavoidably Limit Product Release from a Protein Kinase

Incorporation of divalent metal ions into an active site
is a fundamental catalytic tool used by diverse enzymes. Divalent
cations are used by protein kinases to both stabilize ATP binding
and accelerate chemistry. Kinetic analysis establishes that Cyclin-dependent
kinase 2 (CDK2) requires simultaneous binding of two Mg2+ ions for catalysis of phosphoryl transfer. This tool, however, comes
with a price: the rate-acceleration effects are opposed by an unavoidable
rate-limiting consequence of the use of two Mg2+ ions by
CDK2. The essential metal ions stabilize ADP product binding and limit
the overall rate of the reaction. We demonstrate that product release
is rate limiting for activated CDK2 and evaluate the effects of the
two catalytically essential Mg2+ ions on the stability
of the ADP product within the active site. We present two new crystal
structures of CDK2 bound to ADP showing how the phosphate groups can
be coordinated by either one or two Mg2+ ions, with the
occupancy of one site in a weaker equilibrium. Molecular dynamics
simulations indicate that ADP phosphate mobility is more restricted
when ADP is coordinated by two Mg2+ ions compared to one.
The structural similarity between the rigid ADP·2Mg product and
the cooperatively assembled transition state provides a mechanistic
rational for the rate-limiting ADP release that is observed. We demonstrate
that although the simultaneous binding of two Mg2+ ions
is essential for efficient phosphoryl transfer, the presence of both
Mg2+ ions in the active site also cooperatively increases
ADP affinity and opposes its release. Evolution of protein kinases
must have involved careful tuning of the affinity for the second
Mg2+ ion in order to balance the needs to stabilize the
chemical transition state and allow timely product release. The link
between Mg2+ site affinity and activity presents a chemical
handle that may be used by regulatory factors as well as explain some
mutational effects.