The following information is provided by the hard work and research by authors Todd Weaver, Mason Lees, and Leonard Banaszak. The article "Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site" is the primary reference.[1]Fumarase is a tetramer enzyme involved in the hydration of the substrate fumararate into malate. The enzyme can also catalyze the reverse reaction by dehydrating malate. Fumarase is an essential enzyme in human beings as it is involved in both mitochondrial and cytosolic functions within the cell, regulating the Krebs cycle and amino acid synthesis, respectively. Due to its importance, scientists have actively been characterizing the fumarase. Until 1997 however, little was known about the active until authors Todd Weaver, Mason Lees, and Leonard Banaszak published their research paper titled, "Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site". In this article they sought to find the active site by inducing mutations into fumarase and observe the results.
The fumarase used in this study had been derived from E. Coli, however, the authors used this enzyme because of its convenience and extensive homology that it shares with eukaryotic fumarase. Scientists were unsure about the possible activation site since two locations had often bound substrates. These active sites were described by their primary interacting sidechain, H188 () for site A and H129 for site B. Site A had been seen to bind the substrates citrate and pyromellitic acid while site B had often bound L-malate in the native crystals and β-trimethylsilyl maleate. With there being different substrates binding to different locations, the authors had to select from the two what was the true active site. They suspected site A had been the primary active site since no monomeric form of fumarase has ever been found, the A site was formed by residues from three of the four subunits, and citrate being a known competitive inhibitor of fumarase. Additionally, was a "deep pit" removed from the bulk solvent and it contained a tightly held water molecule (2.5 angstroms) with the imidazole ring in H188.
had been found much closer to the surface of the enzyme and the H129 was found to be the only basic group close to the ligand bound at the B site. This fact had contributed to the issue of activation site identification since biochemical data had claimed that a histidine side chain was one of the bases involved in catalysis. The authors sought out the answer to this uncertainty by mutating H188 () and H129 () into an asparagine residue. The removal of the crucial basic histidine should have reflected in the loss of activity for the enzyme.They had used E. Coli as fumarase producers for the experiment. PCR had been used to amplify the wild-type and mutant recombinant DNA. From there, activity had been measured in order to determine the effect of the mutations on catalysis.
The data had strongly suggested that site A (H188) had been the active site for fumarase since the H188N mutant () had an average activity of 9.62 (μ/ml) and a specific activity of 0.55 (μ/mg). The wildtype had been much more productive with an average activity 4920 (μ/ml) and a specific activity of 116.2 (μ/mg). The wildtype had been most similar to the H129N mutant since it had an average activity of 2080 (μ/ml) and 143.7 (μ/mg) specific activity. The data clearly pointed to site A as the active site.
The authors had then pointed to the crystal structure of site A and B as possible explanations for the activity differences. Site B was found to be a single subunit of the tetramer that includes the residues R126, H129, N131, and D132. Again, H129 was the only possible base in these four residues. The H129N mutant greatly reduced the hydrogen bonding between R126 and the 129th amino acid (originally histidine).In the non-mutated B site, the Histidine played a large role in intermediate stabilization by bonding by using its nitrogen. Further, the asparagine mutant takes away hydrogen bonds from the substrate by hydrogen bonding to the N131 and D132 residues.
Site A had been made up of the H188d (letter denotation dictates which subunit), N326c, K324c, N141b, T100b, S98b, and E331c. The water molecule located at this site interacts with N141b, T100b, S98b, H188d, and the substrate citrate (when available). The tightly bound water molecule (W-26) is shifted 0.7 angstroms in the mutant. This shift is due to the two hydrogen bonds that are now present in the N188d mutant. T100b loses its hydrogen bond due to this change. The alteration of W-26 then affects the binding capabilities of citrate because the substrate relies on a hydrogen bond with W-26 to stabilize the intermediate.
Overall, there were only small changes in conformation of the active sites due to the mutations. Site A relies on the presence of the H188 and in turn the H188 is reliant on the water molecule and E331 for stabilization (helps with basicity). The water molecule could then be potentially involved in the removal of the C3 of L-malate. "The mechanism proposed for H188 is then that it assists in the binding of the C4 carboxylate group of the substrate through Coulombic interactions, and activation of the W-26 to facilitate proton removal from the C3 position."
