[1]Fumarase is a non-iron containing enzyme that catalyzes the hydration/dehydration reaction between L-malate and fumarate. There are two groups that are responsible for the catalytic process of this reaction. The first, is responsible for the removal of a proton from the C3 position of L-malate, which stabilizes the carbanion through a aci-carboxylate intermediate at C4. The second group, gets protonated, and with the removal of –OH from C2, water is formed. Crystallographic studies of fumarase by Weaver & Banaszak in 1996 revealed two different potential active sites in this protein, A-site and B-site. The A-site was made up of three subunits and the B-site was made up of just one. A-site is fixed in a relatively deep pit within the enzyme and had a water molecule that was hydrogen bonded to H188 with a bond length of 2.5 Ᾰ. B-site is more exposed than A-site on the enzyme. A-site and B-site are linked by residues 131-140 which make a one-turn π-helix. The only basic residue that is close to a ligand in B-site is H129. In order to study these two potential active sites and to reveal which is the real active site, Weaver et al. in 1997 changed histidine residues (H188 for A-site and H129 for B-site), the most important residues in the catalytic process, to asparagine. This study revealed that A-site was indeed the catalytic active site.
