Sandbox Wabash 09 Fumarase

Fumarase C, which is found in E. coli, is a enzyme homologous with some eurkaryotic enzymes found in both the cytosol and mitochondria. Fumarase catalyzes the dehydration of L-malate to form fumarate, as well as the reverse hydration reaction. Two basic groups play a role in the overall catalytic process of fumarase. The first basic group is responsible for the removal of a proton from the C3 carbon of L-malate, forming a carbanion. The carbanion is stabilized by an aci-carobxylate intermediate which is formed at C4. The carboxyl group on C4 has a negative two formal charge, stabilizing the substrate after the removal of the proton. The second basic group is protonated, for the creation of fumarate, and leads to the formation of a water molecule as a hydroxyl group is removed from C2. Interestingly, fumarase contains two possible sites at which catalysis occurs, prompting researchers to determine which of the sites is the true active site of the enzyme (Weaver et al., 1997).

You may include any references to papers as in: the use of JSmol in Proteopedia ^[1] or to the article describing Jmol ^[2] to the rescue.

Determining the Active Site

The two possible active sites of fumarase that were studied to determine the actual active site are termed the A-site and the B-site. The A-site is comprised of residues from three of the four subunits, while the consists of residues only from one chain. No active monomeric fumarase has been found, and this was one of observations supporting the A-site. Another observation was the fact that citrate, a known competitive inhibitor of fumarase, bound to fumarase at the A-site. Unlike the A-site, however, the B-site is closer to the surface of the enzyme and not in a deep pit containing an unknown water molecule. The two sites are actually linked by a single subunit at residues 131-140, and there are some important interactions that occur between the B-site and a ligand.

Prior to this research, it had been known that a histidine side chain was one of the bases that took part in catalysis (Brant et al., 1963). Due to this, the residues H129 and H188, at the B-site and A-site respectively, were chosen to test whether the mutation of one would significantly affect the kinetics of the catalysis. Before kinetic analysis, the entire gene of the two mutant forms was sequenced to make sure the intended mutations, H129N and H188N, were the only ones present.

Little change was observed in the specific activity of the , 143.7 micrograms/milligram, as compared to wild type fumarase, 116.2 micrograms/milligram. Meanwhile, the greatly reduced the specific activity to 0.55 micrograms/milligram. The specific activity is roughly two hundred times less than both the WT fumarase and the H129N mutant. Based on this, it was shown that the active site of fumarase is near H188, the A-site (Weaver et al., 1997).

Structural Highlights of the Active Site

On the of fumarase, there are several key residues that are crucial for catalysis. E331 forms a hydrogen bond with H188, which is thought to increase the basicity of the imidazole ring in the histidine residue. H188 then forms a hydrogen bond with the water molecule, that was noted to be present in the active site above. This allows it to interact with the C3 proton on the L-malate substrate and serve as the first basic group in the reaction. (Weaver et al., 1997). The aci-carboxylate formed is then stabilized by various residues, including H188, at O3 and O4. The C4 carboxylate particpates in hydrogen bonding with T187 and N141 while the O4 oxygen participates in hydrogen bonding with H188, N326, and K324. This study reaffirms the hypothesis that a water molecule plays a significant role in the catalysis reaction and is also activated by the ion pair, H188:E331(Weaver et al., 1997).

References

^[3]