Quantifying bond distortions in transient enzyme species by a combination of density functional theory calculations and time-resolved infrared difference spectroscopy. Implications for the mechanism of dephosphorylation of the sarcoplasmic reticulum Ca2 +-ATPase (SERCA1a)

Abstract

The sarcoplasmic Ca2+-ATPase (SERCA1a) forms two phosphoenzyme intermediates during Ca2+ pumping. The second intermediate E2P hydrolyzes rapidly, which is essential for the rapid removal of Ca2+ from the cytosol of muscle cells. The present work studies whether a weakening of the scissile PO bond in the E2P ground state facilitates dephosphorylation. To this end, the experimentally known vibrational spectrum of the E2P phosphate group was calculated with density functional theory (DFT) using structural models at two levels of structural complexity: (i) Models of acetyl phosphate in simple environments and (ii) ~150 atom models of the catalytic site. It was found that the enzyme environment distorts the structure of the phosphate group: one of the terminal PO bonds is shorter in the catalytic site indicating weaker interactions than in water. However, the bond that bridges phosphate and Asp351 is unaffected. This indicates that the scissile PO bond is not weakened by the enzyme environment of E2P. A second finding was that the catalytic site of the E2P state in aqueous solution appears to adopt a structure as in the crystals with BeF3−, where the ATPase is in a non-reactive conformation. The reactant state of the dephosphorylation reaction differs from the E2P ground state: Glu183 faces Asp351 and positions the attacking water molecule. This state has a 0.04Å longer, and thus weaker, bridging PO bond. The reactant state is not detected in our experiments, indicating that its energy is at least 1kcal/mol higher than that of the E2P ground state.