==Alkaline Phosphatase Overview and Questions==

Alkaline phosphatase is an enzyme commonly used in molecular biology research to hydrolyse phosphates from the ends of macromolecules (proteins, DNA, RNA) and nucleotides. This protein is a metalloenzyme (metal containing) that utilizes two zinc atoms and a magnesium atom as cofactors in the active site to carry out catalysis. We will be utilizing a mammalian version of this enzyme to study enzyme function and kinetics in a future lab. Look at the jmol representation of alkaline phosphatase (window on the right, click on "popup" to see a larger version of this representation). The default image is the entire dimeric enzyme complex (containing two identical subunits). Deselect subunit 2 from the right-hand options menu. This will clarify the picture somewhat, displaying only one subunit. Notice that this protein is rich in alpha-helices, but also has a large beta-pleated sheet buried within the interior. The active site is located towards the surface of the protein in a broad groove. The metal cofactors (Mg++ and Zn+) are marked in red, the phosphate ligand (PO42-) in yellow, and the active site amino acids (aa) in teal.

• 1. Knowing that this enzyme functions to remove phosphate from a wide array of biological molecules, including protein, DNA, RNA and nucleotides, why do you think the active site is located near the surface of the protein?

• 2. What role do you think zinc and magnesium ions are playing in active site function of this enzyme? (hint; zinc and magnesium are doing slightly different things here…take a look at the active site structure:

This image shows the active site residues, cofactors and phosphate only. Magnesium ions are , phosphate green, and active site residues colored according to which ligand they interact with. You can identify which metal is which by clicking on the appropriate boxes on the right side menu—“Mg site”to see magnesium labeled, “Zn1 site” to see Zn1 labelled, etc. Note also that dashed lines indicate hydrogen bonds or ionic bonds between amino acids, metal ions and/or phosphate).

Follow the instructions from question #2 to get to the active site image. In the active site image, make sure that “All Sites” is checked, along with “interaction” and “residues”. Now, check the box next to “phos site”. You should now see amino acid residue Arg 166 labelled.

• 3. What function is this residue playing in this enzyme? What do you think would would happen if this amino acid was mutated to glycine, or some other nonpolar small amino acid? Why?

References