Crystallographic studies on shrimp alkaline phosphatase

• Main Author: Backer, M.M.E. (Maaike Maria Eva)
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: Utrecht University 2003
• Subjects: Scheikunde; phosphatase; metalloprotein; zinc triad; metal exchange; x-ray diffraction; synchrotron; multiwavelength anomalous dispersion; MAD; single wavelength anomalous dispersion; SAD; cold adaptation; electrostatic surface potentials; Arctic
• Online Access: https://dspace.library.uu.nl/handle/1874/2620

The earth has large cold areas, such as mountains, oceans and (ant)arctic regions, in which organisms have evolved to survive. This adaptation happens at a molecular level. The question is, how do proteins adjust such that they function at low temperatures? "Cold-active" or "cold-adapted" enzymes have been found to be more efficient at low temperatures and less stable than enzymes from organisms living at moderate temperatures (such as mammals). In this study, the structure of a cold-active enzyme, shrimp alkaline phosphatase (SAP), was determined by x-ray crystallography. It was compared to the structure of a human homologue in order to identify features that explain the adaptation to lower temperatures. This comparison suggests that structure instability of SAP can partly be explained by a reduction in stabilizing interactions, such as fewer hydrophobic residues in the core and lower arginine content. The structure also reveals remarkable differences in charge distribution; it appears that SAP has optimized electrostatic surface potentials in order to attract the substrate, which may increase catalytic efficiency. Alkaline phosphatases contain two zinc ions and one magnesium ion for optimal activity. SAP was however fully occupied with zinc and displayed low residual activity. Crystallographic experiments have shown that one zinc ion can be replaced with magnesium, which seems to happen in conjunction with a conformational change of a ligating histidine. In addition, ligand binding studies have shown that an active site arginine exists in two conformations, which either interacts or does not interact with the bound inhibitor. This observation may have implications for the efficiency of the enzyme. In a conventional single/multiple wavelength anomalous dispersion experiment data are collected at wavelengths near the absorption peak of the element of interest. This method can be simplified by collecting highly redundant data at one "remote" wavelength, where anomalous signals are very small. This technique can ...