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

Journal Article · Tue Aug 14 00:00:00 EDT 2012 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/ja304419t· OSTI ID:1052880

Jacobsen, Douglas M. ^[1]; Bao, Zhao-Qin ^[1]; O'’Brien, Patrick ^[1]; Brooks, Charles L. ^[1]; Young, Matthew A. ^[1]

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1. Univ. of Michigan, Ann Arbor, MI (United States)

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.

• Research Organization: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: USDOE
• OSTI ID: 1052880
• Journal Information: Journal of the American Chemical Society, Vol. 134, Issue 37; ISSN 0002-7863
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: ENGLISH