SiemensExecSummary
- 1. Functional Connections in the Active Site of Alkaline Phosphatase: Interactions between D101 and the Mg2+ ion The project focuses on gaining a broader understanding of enzymes, specifically those belonging to the Alkaline Phosphatase superfamily of enzymes. Enzymes are formed of a string of amino acids that fold into a three dimensional structure; the folded structure is capable of increasing the rates of chemical reactions. Enzymes are present in cells; some chemical reactions important for everyday life would take billions of years to occur without enzymes in solution. Scientific literature in the field has indicated that there are various energetic relationships between different parts of the active site of the Alkaline Phosphatase enzyme. Specifically, there is a connection (energetic coupling) between the Magnesium 2+ (Mg2+) ion and the Aspartate 101 (D101) amino acid within the active site that links the functions of the two components when the enzyme is catalyzing a reaction. Our work aims at dissecting different models that explain this relationship; in other words, the various different amino acids/residues in the active site that could potentially connect the Mg2+ and D101. The methods used to test this concept involved mutating the AP enzyme - numerous mutations of various amino acids were made in different combinations. These different combinations were then expressed in cells, and the enzyme was purified. Upon purification, the various mutations of the enzyme were tested, the activities were measured, and the differences in reaction rates were compared between the mutants. After extensive testing and comparison of the reaction rates of various mutants, we concluded that the Magnesium 2+ ion and D101 amino acid were connected through another amino acid, Aspartate 51 (D51). Although our data does not conclusively prove this link to be true, all the results are consistent with this model, and support the connection through D51.