Price To Be Paid for Two-MetalCatalysis: Magnesium Ions That Accelerate Chemistry Unavoidably LimitProduct Release from a Protein Kinase

摘要

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 andthe cooperatively assembled transition state provides a mechanisticrational for the rate-limiting ADP release that is observed. We demonstratethat although the simultaneous binding of two Mg²⁺ ionsis essential for efficient phosphoryl transfer, the presence of bothMg²⁺ ions in the active site also cooperatively increasesADP affinity and opposes its release. Evolution of protein kinasesmust have involved careful tuning of the affinity for the secondMg²⁺ ion in order to balance the needs to stabilize thechemical transition state and allow timely product release. The linkbetween Mg²⁺ site affinity and activity presents a chemicalhandle that may be used by regulatory factors as well as explain somemutational effects.