Fumarase (fumarate hydratase) catalyzes the hydration of fumarate (double bonded) to form malate. The hydration reaction, which includes a carbanion transition state, has OH- addition occur before H+. Fumarase can be found in the urea cycle, an important biochemical reaction used to produce urea. [1] Fumarase C enzymes are tetrameric, non-iron containing, and are specificially found in E. coli. They are known to be similar to mitochondrial enzymes found in eukaryotic cells.[2]
Debate
of fumarase C from E. coli have been made using PCR and recombinant DNA. Two different carboxylic acid binding sites (A+B) were observed in the crystal structures of the WT inhibited forms of the enzyme. The H188N mutant has L-malate bound at active site A whereas the has L-malate bound at site B.
Crystallographic studies with several inhibitors including pyromellitic acid and B-trimethylsilyl maleate yielded interesting results. While both inhibitors are related to the normal substrate, each was discovered to be bound at different sites. The binding site of inhibitors citrate and pyromellitic acid was deemed the A site while a second site containing L-malate and B-trimethylsilyl maleate was labeled as the B-site. The first argument for site A being the active site was that the A site was formed by 3 of the 4 subunits. Secondly, citrate was used at high concentrations as a crystallizing agent and is known to competitively inhibit fumarase. As such, in X-ray studies, citrate was not able to be removed readily from the specimen preparation and it pointed to the A site. In regards to the B site, it was first noted that atoms of a single subunit formed the B site. Strong arguments were then made against the B site as no active monomeric form of fumarase has ever been described.
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True Active Site and Structural highlights
In lieu of the fact that a histidine side chain was one of the bases in the catalytic reaction, in order to determine which of the two sites was indeed the active site, histidines could be observed. By mutating the histidines at the two active sites, one would be able to determine that if the A-site was active, changing the would dramatically affect the catalytic activity. Rather, if the B-site was the active site, then a mutation in the would affect catalysis. In support of the A site is the fact that it is the only active tetramer and the specific mutations found that H188N reduced catalytic activity whereas H129N had little to no effect. Furthermore, the crystal structures confirm that water plays a significant role in the catalytic reaction. Specifically, water seems to be located in a precise location to act as a base and remove a proton from L-malate at the C3 position. Additional studies will have to be conducted to determine a more detailed role that water plays in altering catalytic activity of fumarase.
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