Benjamin Washer
Function
Fumarase C is an enzyme found in E. coli (EfumC) that is similar to the cytosolic and mitochondrial enzymes found in eukaryotic cells and is used to catalyze the hydration/dehydration reaction between L-malate and fumarate. Because of its relative simplicity, homology with important eukaryotic enzymes and important metabolic influence, fumarase has been the object of much study. However, upon analysis of fumarase, it was found that there were two "active" sites and much debate has arisen regarding which is the most influential or "true" one. This article describes the debate and the process used to determine the true active site.
Relevance
Structural highlights
The general mechanistic idea about the fumarase reaction is that at the active site, L-malate is converted to fumarate via several steps. First, the a proton is removed at the C3 by a basic residue near the active site forming an aci-carboxylate intermediate. In the second step, the -OH group on the C2 carbon leaves as an OH- ion. This step is thought to be aided by another protonated basic group on the enzyme. However, upon X-ray crystallography analysis of the E-S complex, it was found that there were traces of L-malate at , where His is the proton transfer mediator (The red His referred to as site A and the blue His is referred to as site B). This raises the issue as to which is the the true site. It is thought that while the B site has several stabilizing residues such as , the A site is formed by residues from three of the four subunits are shown here.
Since the biochemical data suggested that a histidie residue was participating in the above reaction, mutations on both H129 and H188 were carried out and crystal and activity measurements were then conducted. Should the A site be the active site, then a mutation on the H188 would greatly affect the catalytic activity of fumarase. The same logic holds for the B site. The PCR based mutations were made using a Perkin-Elmer Thermocycler 480 and were expressed using JM105. It was found through experimentation that mutating the H129 residue did not adversely change the enzyme activity. However, mutating the H188 residue drastically lowered the enzyme activity (from ~116 u/mg to ~0.55 u/mg) and gave the final piece of evidence needed to determine that the A site was indeed the active site. This decrease in activity is partially due to the change in H-bonding between the solvated water molecule in the deep pocket of the A site and the rest of the active site residues. Helping to stabilize this active site is the which acts by increasing the basicity of H188 and this relay effect is in turn passed to the active water site. This increase in basicity of H188 and increased effectiveness of the active water site helps to stabilize the substrate intermediate and promote the proton transfer sequence described above.
