Hydrolysis of the E2P Phosphoenzyme of the Ca2+-ATPase: A Theoretical Study

Dephosphorylation of the E2P phosphoenzyme intermediate of the sarcoplasmic reticulum Ca2+-ATPase was studied using density functional theory. The hydrolysis reaction proceeds via a nucleophilic attack on the phosphorylated residue Asp351 by a water molecule, which is positioned by the nearby residue Glu183 acting as a base. The activation barrier was calculated to be 14.3 kcal/mol, which agrees well with values of 15–17 kcal/mol derived from experimentally observed rates. The optimized structure of the transition state reveals considerable bond breakage between phosphorus and the Asp351 oxygen (distance 2.19 Å) and little bond formation to the attacking water oxygen (distance 2.26 Å). Upon formation of the singly protonated phosphate product, Glu183 becomes protonated. The bridging aspartyl phosphate oxygen approaches Lys684 progressively when proceeding from the reactant state (distance 1.94 Å) via the transition state (1.78 Å) to the product state (1.58 Å). This stabilizes the negative charge that develops on the leaving group. The reaction was calculated to be slightly endergonic (+0.9 kcal/mol) and therefore reversible, in line with experimental findings. It is catalyzed by a preorganized active site with little movement of the nonreacting groups except for a rotation of Thr625 toward the phosphate group.