The enzyme known as fumarase catalyzes the conversion of malate to fumarase. Crystallographic studies using inhibitors revealed that the inhibitors bound to two different locations. This indicated that there were two potential active sites for fumarase. Site A was located within a pit and was made up of atoms from 3 of the 4 subunits present within fumarase. Site B was located towards the surface of the enzyme and was made up of only 1 of the 4 subunits.There locations can be seen here. . Debate about which of the sites was the true active site was centered around the fact that there was no known monomeric fumarase. Since site A was made up of atoms from multiple subunits, site A seemed like the likely active site for fumarase [1]. This was tested by mutating the catalytic His on both of the sites and observing the amount of fumarase activity.
Since it was known that His served as a base within the mechanism, the His within each site was mutated to an Asn in order to see the effect on fumarase activity. A mutation of site A was characterized by a mutation of His 188 and the mutation of site B was characterized by a mutation of His 129. A Nickel column was used to purify each of the mutant sites and SDS-Page was used to ensure that contaminants were not present[1]. Speciifc activity of fumarase in u/mL revealed how much malate had been converted to fumarate in each mutant. The wild type fumarase activity was 4920.0 u/mL. The amount of fumarase activity for the mutation of His 129 was still relatively high (2080.0 u/mL). The activity for the His 188 mutation had fallen close to 0 (9.62 u/mL). This showed that Site A was the true active site because a mutation in His 188 prevented catalysis of the conversion of malate to fumarate. Crystal structures were obtained from each site in order to see the finer details of the mechanism of the reaction as well as the structure of the active site.
The active site of fumarase consists of several significant residues with His 188 being one of the most important. The residues found within the active site can be found here. . The malate intermediate possesses a double negative charge on the C4 carboxylate and is very important for the catalysis of this reaction, but it requires stabilization due to the significant charge. His 188 along with several other residues coordinate the carboxylate on C4 of the malate through hydrogen bonding. For example, T187 and N141 form hydrogen bonds with the O on C3 and H188, N326, and K324 form hydrogen bonds with the O on C4 [1]. This hydrogen bonding coordination allows for malate to be in the correct position for catalysis. The residues that participate in the Hydrogen Bonding can be seen here. . The other function of H188 is that it pairs with E 331 in order to make water more basic [1] . By deprotonating water, water is able to remove a proton from C3 of malate. This creates a positive charge on C3 that will stabilize the intermediate. This positive charge will cancel out the double negative charge on the carboxylate of the C4 atom, making a more stable intermediate. The residues that cooperate to make the water molecule more basic can be found here. .
CFTR
The residues involved in conduction include R117, R334, and R347. These residues can be seen here. . They are located toward the middle of the structure of the protein. These mutations may have caused the CFTR to lose the ability to facilitate movement of Cl- ions in and out of the cell because a change in the shape of the channel itself may prevent chlorine from moving in and out of the cell.
