A program of complementary experimental and theoretical studies is proposed to elucidate the structural chemical basis of the """"""""high energy phosphate bond"""""""". All kinds of metabolic energy exchanges are based on the ATP cycle, in which free energy is stored by condensation of ADP and Pi and then released when needed by hydrolysis of the terminal phosphate of ATP. The proposed research will investigate a series of small, model phosphates that are involved in the ATP cycle and that span the range of free energy of hydrolysis or """"""""phosphate group transfer potential."""""""" The series will range from high-energy phosphoenolpyruvate to low-energy 3-phosphoglycerate and will include inorganic orthophosphate and pyrophosphate species. The experimental studies will be highly accurate X-ray and neutron diffraction analysis of about ten selected phosphate crystal structures. These crystallographic studies will extend to detailed analyses of the thermal vibrations and the electron density distributions in the crystals, and will provide accurate molecular geometries and detailed maps of the valence electron density deformations and molecular electrostatic potentials. The same series of phosphates will be studied theoretically, by ab initio and semi-empirical quantum chemical calculations and by Monte Carlo statistical mechanical simulations of aqueous solvation structures. Experimental and theoretical electron densities and electrostatic potentials will be compared in order to cross-check and calibrate the relative effects of experimental errors and theoretical approximations. Accurate molecular geometries and electron density distributions will reveal the nature of the phosphorus-oxygen bonding and allow, e.g., net atomic charges and bond orders due to variable pPi(O) yield dPi(P) partial double bond character to be deduced. Comparisons of these results for high-energy versus low-energy and for unhydrolyzed versus hydrolyzed phosphates will clarify the roles of ionic charge delocalization and resonance stabilization of electronic structure as determinants of the free energy changes accompanying biological phosphate hydrolyses. The important role of hydration of the hydrolysis reactants and products will be studied using the charge density distributions and electrostatic potentials to calculate electrostatic interaction energies for phosphate ion-water molecule association interactions and to parameterize potential functions for statistical mechanical simulations of solvation structures for biological phosphates in aqueous solution.
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