This is an accepted version of a paper published in Journal of Physical Chemistry B. This paper has been peer-reviewed but does not include the final publisher proofcorrections or journal pagination.Citation for the published paper: Rudbeck, M., Nilsson Lill, S., Barth, A. (2012) "The influence of the molecular environment on phosphorylated amino acid models: A density functional theory study" Journal of Physical Chemistry B, 116 (9)

The infrared spectrum of phosphoenol pyruvate (PEP) in aqueous solution was studied experimentally and theoretically in its fully ionized, singly protonated and doubly protonated form. The density functional theory with the B3LYP functional and with the 6-31G(d,p), 6-31++G(d,p), and 6-311++G(d,p) basis sets were used in the theoretical study. The calculations with the two latter basis sets and the CPCM continuum model for water showed good agreement with the experiments except for vibrations assigned to hydroxyl groups. These needed to be modeled with explicit water molecules. The effects of deuteration and of (13)C(2,3) labeling of PEP were reproduced by the calculations.

The addition of extravalence, polarization and diffuse functions, were studied in order to conclude how they affect the PAO stretching frequencies of several biological relevant phosphate molecules. The results show that the polarization and the diffuse functions have opposite effects on the frequencies: the polarization functions downshift while the diffuse functions upshift the frequencies. The effect of the valence functions was more difficult to interpret. The effect of the conductor-like screening model (CPCM)-continuum model was also studied. The results show that the CPCM-continuum model has a substantial effect on the frequencies for these small molecules. The continuum model's efficiency is mainly due to its effect on the geometries and not on the frequencies.

Dephosphorylation of the E2P phosphoenzyme intermediate of the sarcoplasmic reticulum Ca(2+)-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.

Alpha-carboxy-4-nitrobenzyl phosphate 4 and its derived monomethyl phosphate ester 5 were synthesized and purified by anion-exchange chromatography. A gradient of LiCl was necessary for elution of the anion-exchange column to avoid unexpected thermal decarboxylation that occurred during vacuum evaporation when the volatile triethylammonium bicarbonate buffer was used. Photolysis of each compound was accompanied by decarboxylation, and 4 released inorganic phosphate with near-100% stoichiometry. Time-resolved infrared spectroscopy of the photolysis reaction, coupled with density functional theory calculations of vibrational frequencies, enabled us to infer a mechanism for the photolytic pathway, although there was some evidence for a second pathway also being operative. In contrast to the results for 4, photolysis of 5 appeared to release little or no monomethyl phosphate.