Determining the Active Site of Fumarase- Austin Dukes
In studying Fumarase C from E. coli, an enzyme that catalyzes the hydration/dehydration reaction between L-malate and fumarate, Weaver, Lees, and Banaszak were looking to locate the active site[1]. It is thought that the catalytic process involves two basic groups: one that helps to remove a proton from the C3 of L-malate, and another that would be responsible for being protonated and making the leaving of OH- from C2 more favorable. Different inhibitors related to the usual substrate were studied, and it was found that they bound at two different sites on the enzyme with basic subunits: A and B. This forced the researchers to do more work in order to determine which site is the active site. That being said, they felt confident they would discover it is A: [2] is made up of atoms from 3 of the 4 subunits of fumarase and located deep inside the enzyme, whereas is only made up of 1 subunit and is located on the outside of the enzyme. Since there is no known active monomer of fumarase, it seemed more likely that A would be the active site.
Due to its pH/temperature dependence, it seemed logical that the active site of fumarase would include a histidine residue. This exists in both sites: at site A, forms a short hydrogen bond with water, whereas at site B, is the only basic group near where the ligand was found to bind. It makes sense then to alter these sites by changing H188 and H129 to another residue that will not be as basic as histidine (in this experiment, they choose asparagine). If H188N severely alters the catalytic activity of fumarase, it is likely that A is the active site; if H129N severely alters the catalytic activity of fumarase, it is likely that B is the active site.
From running this experiment, the researchers determined no significant difference between the specific activity of the wild-type fumarase (116.2 +/- 14.0 µ/mg) and the mutant (143.7 +/- 10.0 µ/mg). Conversely, the mutant displayed a sharp drop in specific activity (0.55 +/- 0.044 µ/mg). Because the H188N mutant had such a drastic impact on specific activity, it now seems clear that site A is the active site.
In the active site of fumarase, there are several important residues; that being said, the most vital are a water molecule , which act as the 2 aforementioned bases. The presence of H188, which participates in a short hydrogen bond with W-26, makes the water a stronger base. As a result, the water gets protonated by the C3 of the substrate, L-malate. When W-26 is protonated, it is more able to stabilize the double-negative charge on the aci-carboxylate at the C4 along with several other residues in the active site such as H188, T187, N141, N326, and K324. By stabilizing the reaction intermediate as such, fumarase is an effective enzyme catalyst. That being said, it is worth noting that the enzyme still functioned at a minimal rate in the absence of H188. This is likely because in the proposed mechanism, W-26 would still be able to protonate C3 of the substrate in the absence of H188, although it would be less favorable. As a result, W-26 seems to be the more important of the two bases involved.
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