Sandbox Wabash 05 Fumarase

From Proteopedia

The Active Sites of Fumarase

is an enzyme that catalyzes the hydration/dehydration reaction that converts fumarate into malate and vice versa. After extensive experimental studies on this particular enzyme, researchers discovered fumarase has two sites that are involved in carboxylic acid binding. These sites are known as the A-site and the B-site. These two sites are both composed of hisitdine residues that act as the basic group to interact with carboxylic acids and water in their respective binding sites. However, during initial discovery of the two sites, a question arose about which site acted as the active site for the enzyme and catalyzed the fumarate to malate reaction. Weaver, Lees, and Banaszak were able to determine that the A-site is the true active site of fumarase by implementing mutations on each of the sites by using E.Coli and performing PCR using recombinant DNA. The A-site histidine, which is involved in binding, is and the B-site histidine is . The experimenters expressed mutations on each of these histidine residues in separate cellular cultures by . They then observed how the specific activity changed for each mutated enzyme. They found that the asparagine substitution for histidine at the A-site resulted in a 200-fold decrease in specific activity of the fumarase as compared to the wild type. However, the mutation at the B-site resulted in a minimal effect on specific activity as compared to the wild type. Therefore, they concluded that the A-site, which includes HIS-188, must be the and that the B-site does not have a significant role in the catalytic ability of the enzyme ^[1].

Active Site and Stability

The fumarase active site is stabilized by several interactions. First of all, it is important to note the amino acid residues that reside in the active site. These residues are HIS-188, THR-100, SER-98, ASN-141, and GLU-331. As previously stated the HIS-188 is important in carboxylic acid binding. Furthermore, the active site also contains a water molecule that is important for stabilization. The water molecule within the active site allows for extensive hydrogen bonding among the SER-98, ASN-141, THR-100 and HIS-188 residues, which stabilizes the active site of the fumarase enzyme. The water molecule further acts as a stabilizer in regards to the intermediate of the L-malate. The intermediate, known as the aci-carboxylate intermediate, has a double negative charge, which is stabilized by the water molecule that is in the active site because it acts as a base. These hydrogen bonds orient the C3 and C4 carbons of L-malate in the active site correctly in order for catalysis to occur. Lastly, the GLU-331 forms hydrogen bonds with the HIS-188 and water in order to increase the basicity of the HIS-188 and in turn the basicity of water. This water removes the proton from the C3 of the malate and is essentially an important piece of the catalytic mechanism. ^[2].

References

1,2. Weaver, Todd, Mason Lees, and Leonard Banaszak. "Mutations of Fumarase That Distinguish between the Active Site and a Nearby Dicarboxylic Acid Binding Site." Protein Science 6.4 (2008): 834-42. Pub Med. Web.