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Theoretical Analysis of the Metal Substitution and Water Structure in the Active Site of Phosphotriesterase



Morris Krauss, L Olsen, J Antony, L Hemmingsen


Theoretical examination of the cadmium substitution in the native zinc phosphotriesterase provides insight into the coordination binding and emphasizes the importance of waters hydrogen-bonded to ligands in the active site and bound to the metals to both the structure and mechanism. The native ZnZn and cadmium substituted Zn-Cd and Cd-Cd structures are optimized with effective fragment potentials (EFP) providing a realistic representation of the protein environment surrounding the active site. One to three waters are included in the active site in addition to the bridging hydroxide. These waters significantly affect all aspects of the structure of the active site including the metal-metal distance and the coordination binding of the first shell aspartate. Comparison of the predicted structures with recent x-ray results on these systems is hindered by the appearance of ethylene glycol molecules in the x-ray structure of the active site. We suggest that the ethylene glycol required for crystallization distort the active site structure and the theoretical results provides additional insight into the solution structure. In order to validate the predicted water structure in the active site, the NMR Cd113 shift is calculated from the theoretical structures and compared to the experimentally observed shifts. The calculated NMR shifts are correlated to the coordination number at the site. The ZnCd enzyme has to have a six-coordinate Cd site. This still leaves an ambiguity in the assignment addressed by the theoretically optimized structures.Although at least three waters bind in the active site, optimizing the structures with less than three provides insight into the effect of water binding on the active site structure. There are two possible ZnCd structures and the most stable theoretical structure is Cd1Zn2 with the metal bound to the Od1 of the carboxylate of the first-shell aspartate designated M1. However, the energy difference between Cd1Zn2 and the lowest energy Zn1Cd2 structure is less than 1 kcal/mol and decreasing with the addition of waters. Theoretically a number of energetically close water structure are found for the CdCd structure. Formally, one of these agrees with the x-ray structure and is supported by the NMR assignment. The theoretical structures suggest that a number of different hydrogen-bonded arrangements have close energies and the arrangement in solution may represent an average for both ZnCd and CdCd enzymes.
Journal of the American Chemical Society


113Cd NMR shift, active site optimization, effective fragment potentials, metal substitution, phosphotriesterse


Krauss, M. , Olsen, L. , Antony, J. and Hemmingsen, L. (2008), Theoretical Analysis of the Metal Substitution and Water Structure in the Active Site of Phosphotriesterase, Journal of the American Chemical Society (Accessed March 4, 2024)
Created October 16, 2008