When studying fumarase 2 sites were identified to potentially be the active site. The first site (A-site) binds inhibitors (citrate, and pyromellitic acid) and is made up of parts of 3 of the 4 subunits that comprise fumarase. The second site (B-site) binds L-malate and is made up by parts of 1 of the 4 subunits. The A-site is located deeper within the enzyme complex and binds water that interacts with whereas the B-site exists closer to the surface and the only basic group it binds is . [1]. In order to differentiate between the sites to determine the true binding site scientists compared whether changing Histidine 129 or Histidine 188 impacted the complex. Histidine was picked because previous research demonstrated that histidine was participating in catalysis. The researchers then inserted mutations (changed the histidine to asparagine) and determined that the A-site was the active site based on mutation results and crystal structures. The H188 mutation dramatically reduced enzyme activity whereas the H129 mutation had almost no effect. The A-site coordinates H-bonds utilizing , which serve to position the C3 and C4 atoms of in the complex. Finally the A-site also activates Tryptophan to remove a proton from the C3 position, leading to the activation of a water molecule.
