User:Jeremiah C Hagler/Sandbox 1

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). This is a cartoon representation of the secondary structures found in this protein. Alpha helices are in red, beta pleated sheets in yellow. 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 are represented as balls (green for Mg2+ and grey for Zn2+), the phosphate ligand (PO42-) in orange (phosphorous) and red (oxygen), 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, metal cofactors and phosphate only. The magnesium ion is green, zinc grey, phosphate orange (phosphorous) and red (oxygen)and the active site residues colored according to their make up (carbon light grey, nitrogen blue, oxygens light green or red). You can identify the various components of this image by hovering the mouse cursor over the appropriate component--a name will pop up, along with a number indicating it's position, etc. Note also that thick lines indicate covalent bonds, the thinner lines are ionic bonds between amino acids, metal ions and/or phosphate. Note that hydrogen bonds are not shown in this image.

In the active site image, move your cursor around to find the amino acid residue Arg 166.

• 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