Sandbox Wabash 27 Fumarase

Fumarase C (fumarate hydratase) is an enzyme found in eukaryotic cells which catalyzes the reaction between L-malate and fumarate.^[1] The catalysis proceeds via the deprotination of the C3 carbon of L-malate, which is followed by the loss of the -OH group attached to the C2 carbon; the intermediate for which is a carbanion transition state.^[2] In order for this catalysis to occur L-malate must be bound to fumarase. Studies have determined that fumarase has two carboxylic binding sites (site-A and site-B) which could potentially be the active site for the conversion of L-malate to fumarate. Mutations of these sites were performed in order to determine their effects on the enzymatic activity of the fumarases. Specifically, the key histidines which were known to play an active role in the binding process were removed for each respective site. The results showed that a mutation of site-B lead to no statistical difference in specific activity when compared to wild type activity, while site-A's mutation lead to a significant decrease in enzyme activity. Thus, it was determined that binding site-A is in fact the active site for the conversion of L-malate to fumarate ^[3].

Binding Site Characteristics

Site-A:Site-A can be described as being comprised of atoms from three of the four sub-units (tetramer). It has been shown to be in a deep pit which is fairly removed from solvent, while also containing a bound water molecule. The bound water molecule is a key feature of this site, as it may act as the proton acceptor in the first step of the catalytic reaction. The residue of interest, H188, interacts with this water molecule.

Site-B:Site-B can be described as being comprised of residues from only one sub-unit (monomer). Its main interactions stem from residues 131 to 140, which is just more than one turn of a π-helix. Notable residues that provide stabilization include D132, N131, H129, and R126; H129 is the only basic group close to a ligand bound at this site, making it the residue of interest for mutation.

Mutations were performed on both histidines of interest (H188, H129) in site-A and site-B respectively, and the crystal structures of each were analyzed. Each of these were replaced by asparagine in order to determine the site’s role in the enzymatic catalysis of L-malate^[4].

H188N: Effects of Mutation

H188N (mutation of H188 in fumarase) replaced the H188 residue with N188 located in site-A. In the wild type (WT) fumarase H188 was shown to interact with the surrounding residues (S98, T100, N141) as well as with the crystalizing agent (citrate). However, with N188 replacing H188, the citrate was no longer observed to be interacting with the residues, which suggests that H188 is critical for the binding of the substrate to the enzyme at site-A. H188N was shown to create no effect on site-B as H188 is not present in this site, suggesting that site-A is the active site. Furthermore, the specific activity of the wild type sample was measured to be 200 times more than that of the H188N sample (116.2 and 0.55 μ2/mg2). This massive decrease in activity is representative of site-A being the active site of fumarase^[5].

H129N: Effects of Mutation

H129N (mutation of H129 in fumarase) replaced the H129 residue with N129 located in site-B. In the WT fumarase H129 was show to interact with residues as a single sub-unit (D132, N131, R126) with no apparent binding to citrate. When N129 replaced H129 citrate is still not observed as binding at site-B, therefore suggesting that site-B is not the active site of fumarase. N129 was observed to reduce the binding affinity of ligands by creating new bifurcated hydrogen bonds with N131 and D132. Also, it was determined that the specific activity of site-B was not found to be significantly different than the WT (143.7 and 116.2 μ2/mg2) further suggesting that site-B is not the active site. To further confirm that H129N did not bind with ligands at site-B, a crystal was soaked in β-trimethysilyl maleate (TMSM), which still did not produce any signs of binding at the site^[6].

Summary and Conclusions

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