Fumarase is enzyme found commonly in E. coli that catalyzes the hydration and dehydration reaction between L-malate and fumarate. Its homology to other mitochondrial enzymes found in eukaryotic cells has allowed researchers to make predictions and other observations of this tretrameric enzyme [1]. In the catalytic process this particular enzyme is accredited for is believed to occur between the combination of two basic groups. The first group is responsible for deprotonating from the C3 position of L-malate, which causes a stabilized carbanion intermediate at C4. In the second, now basic group, it is protonated and the removal of –OH forms a water molecule [2].
With the help of crystallographic studies on wild-type fumarase researchers have found some unusual characteristics when observed with different inhibitors. With the use of pyromellitic acid and beta-trimethylsilyl maleate two different activation sites seemed to appear. The original activation site (A-site) is composed of atoms from three to four subunits and was determined by the binding site of the inhibitors citrate and pyromellitic acid. The second activation site (B-site) was identified by the use of beta-trimethylsilyl maleate and was formed by atoms of a single subunit. These two findings ultimately led to the confusion as to which two sites accepting the inhibitors was the true active site.
In order to test this dilemma, scientists used mutations at both proposed active sites. At A-site a mutation at H188N () was used, and at B-site a mutation at H129N ( )was used. Histidine was the primary residue previously observed to be involved in the catalytic activity of fumarase, which was why these particular residues became the focus of the mutation [3]. These mutations resulted in a significant decrease of activity for fumarase when the H188N was altered, but almost no effect when H129N was changed. This extreme difference between activity led to the conclusion that the true active site was A-site [4].
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