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[1]. 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 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 . 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 ( for A-site and His 129 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.
