P-O bond destabilization accelerates phosphoenzyme hydrolysis of sarcoplasmic reticulum Ca2+ -ATPase.

The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+ -ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm(-1). This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 A shorter terminal P-O bonds and a 0.09 A longer bridging P-O bond that is approximately 20% weaker, the angle between the terminal P-O bonds is wider, and -0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P-O bond of E2-P accounts for a 10(11)-10(15)-fold hydrolysis rate enhancement, implying that P-O bond destabilization facilitates phosphoenzyme hydrolysis. P-O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins.