Sandbox Wabash 01 Fumarase

Fumarase C from E.coli is an enzyme that catalyzes the reversible hydration/dehydration reaction between the metabolites L-malate and fumarate (). Fumarase C is an tetramer enzyme found in a large variety of organisms namely eukaryotes, bacteria, yeast, molds, plants, invertebrates, and mammals (Hill, & Teipel, 1971). Due to the enzymes similarity with eukaryotic Fumarases, it has been much easier to characterize and understand its mechanisms. However, the true active site of Fumarase has been up for much debate.

Two sites have been identified as the possible active sites of Fumarase C in E coli, they are designated as the A-site and B-site. The A-site was initially considered as the active site for a couple reasons, 1) the active site of fumarase has never been described as monomeric (which the A-site has multi-subunits), 2) a well known active-site inhibitor of fumarase C known as Citrate, through experiments was logistically found near the A-site of the enzyme (Weaver, Lees, & Banaszak, pg 834-835, 1997). However, the A-site is not found near the surface of the enzyme and has an unusual water attached. His188, a side-chain found in the A-site ()was observed interacting with the water, and also observed hydrogen-bonding with an oxygen atom belonging to our citrate that was mentioned earlier. At the same time however, there was speculation that the active site of Fumarase C may exist at the B site because its closer to the surface of the enzyme (Weaver et. al, pg. 835, 1997). The A site and B site are linked by approximately 9 residues. Within the B site is His 129 () which is the residue closest to the proposed active site where the ligand binds ().

This information led Weaver et al. (1997) to their hypothesis. If the A-site was the active site then mutation of H188 should affect catalytic activity of Fumarase C, but if the B-site is the active site then mutation of H-129 should affect catalytic activity. The experimenter's induced mutations in both sites using PCR reactions changing the Histidine's into Asparagine's ()(). In order to ensure this was done correctly the entire gene was sequenced and they found the only differences in the original gene coding for Fumarase C was the sites they mutated. These mutated strains were purified using Nickel affinity chromatography, and SDS-PAGE. What the results showed was that H-129 (B-Site) had little effect on activity when mutated. When H-188 was mutated it dramatically affects the catalytic properties of Fumarase C, giving the researchers evidence that the active site of Fumarase C is indeed the A-site (Weaver et. al, pg. 836, 1997).