Fumarase C from E. coli catalyzes the hydration/dehydration reaction between the L-malate and fumarate. It belongs to a group of enzymes which are tetrameric with each monomer containing about 460 amino acids. There are two basic groups believed to involve in the catalytic process.The first group is responsible for the removal of a proton from the C3 position of L-malate. The resulting carbanion is stabilized by an aci-carboxylate intermediate formed at C4. The second basic group on the protein is thought to involve in the last stage of the catalytic process. When producing fumarate, the second basic group would be protonated and the removal of the -OH from C2 results in the formation of a water molecule. The proton in the first group has unusual properties and is believed to be removed as the next substrate molecule binds.This enzyme also have an unusual subunit arrangement as it has a core consisting 20 20 alpha helices, 5 from each of the subunits.
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Two Possible Active Sites
The crystallographic studies using pyromellitic acid and beta-trimethylsilyl maleate indicated the presence of two possible binding sites. The original tungstate site, a heavy atom
derivative, is one of the possible sites called A-site. It's found to be a binding site of the inhibitors citrate and pyromellitic acid. This site is comprised of atoms from three of the four subunits and is relatively deep in the structure of the enzyme. However, another crystallographic study identified a second blinding site as it contained L-malate and beta-trimethylsilyl maleate as well. This second possible binding site called B-site is formed by atoms from only one subunit and is relatively closer to the surface of the enzyme. Therefore there was a dilemma about which site is the true active site for fumarase C.
Determination of True Active Site
Research suggested that a histidine side chain was one of the bases participating in the catalytic reaction therefore testing whether HIS 129 or HIS 188 affected catalytic activity offered a way of resolving the two site dilemma. If the A-site was the active site, mutating HIS 188 should greatly change the catalytic activity.On the other hand, if the B-site was the active site then a mutation instead at HIS 129 should affect the catalytic process. To conduct the experiment, two mutant fumarases H129N and H188N were studied using crystal structure analysis and activity measurement. In both mutants, the histidine are mutated to asparagine. Two evidence supporting A-site is the true active site was provided by the experiments. First, the activity result data indicated that H129N (specific activity 143.7 ± 10.0 u/mg) have about the same specific activity as the wild type (specific activity 116.2 ± 14.0 u/mg) while the H188N mutation dramatically affects the catalytic reaction - it's specific activity (0.55 ± 0.044 u/mg) is about 200 times less than that of wild type or HI29N. The crystal structure analysis provided the second evidence. The crystal structure of H129N showed that the mutated protein had essentially the same conformation as the wild type but appeared to dramatically reduce binding of ligands at the B-site while the crystal structure of H188N showed that the absence of the histidine side chain effectively reduces binding of citrate so that it is missing in the electron density maps.
Active Site Structure
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