Mechanistic insights into the phosphoryl transfer reaction in cyclin-dependent kinase 2: A QM/MM study

Abstract

Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

Fig 1. Proposed pathways for the substrate-assisted and base-assisted mechanisms.

Dotted lines represent bonds that are broken or formed during the reaction.

Fig 2. Atoms included in the QM region for QM/MM calculations.

The valences of the atoms lying at the QM/MM boundary were completed with hydrogen atoms.

Fig 3. Potential energy profiles for both, substrate-assisted (black) and base-assisted (red) mechanisms.

The numbers show the relative energy for each stationary point with respect to reactants.

Fig 4. Molecular structures of the stationary points in the substrate-assisted mechanism.

(A) Reactant state. (B) Transition state. (C) Product state.

Fig 5. Molecular structures of the stationary points in the base-assisted mechanism.

(A) Reactant state. (B) Transition state TS1. (C) Intermediate state Int. (D) Transition state TS2. (E) Product state.

Fig 6. Wiberg bond orders and bond order derivatives of the bonds that are broken or formed during the reaction for both mechanisms.

(A) Bond orders for the substrate-assisted mechanism. (B) Bond orders for the base-assisted mechanism. (C) Bond order derivatives for the substrate-assisted mechanism. (D) Bond order derivatives for the base-assisted mechanism. Dotted lines show the position of the stationary points on the reaction coordinate.

Fig 7. Evolution of NPA charges for the most relevant atoms in the reaction.

(A, C and E) Charges for P_γ, PO₃ and Mg²⁺ in the substrate-assisted mechanism, respectively. (B, D and F) Charges for P_γ, PO₃ and Mg²⁺ in the base-assisted mechanism, respectively. Dotted lines show the position of the stationary points on the reaction coordinate.

Fig 8. Reactants structures showing hydrogen bonding interactions (dashed blue lines) that stabilize the γ-phosphate of the ATP molecule.

(A) Substrate-assisted mechanism. (B) Base-assisted mechanism.