Page Created and Authored by Lucas T. Knutson
Crystallographic Determination of the Active Site in Fumarase C from E. coli
Fumarase C is an enzyme from Escherichia coli that catalyzes the stereospecific, reversible interconversion between L-malate and fumarate via the hydrolysis of the carbon-oxygen bond. The hydration/dehydration reaction catalyzed by fumarase C is of particular interest, because it plays an essential role in the metabolic citric acid cycle[1]. Fumarases from eukaryotic organisms have been studied and characterized to a much greater extent, compared to fumarase C from E. coli. In an attempt to further characterize EfumC, crystallographic studies were conducted using the enzyme, however the studies produced some unexpected results. Crystal structures of EfumC revealed that the enzyme has two dicarboxylate binding sites. More specifically, crystallographic studies that utilized inhibitors related to the enzyme’s normal substrate found that the inhibitors pyromellitic acid and β-trimethysilyl maleate bound to two different sites, which were named the A-site and B-site, respectfully[2]. These contrasting results raised the question: which of the two sites is the active site of the enzyme?
In order to answer this question, an experiment that tested each of the sites independently would need to be designed and conducted. Biochemical data suggested that a histidine side chain in both sites – in the A-site and in the B-site – was one of the key factors in substrate binding, so a mutation of the histidine residue in either of the sites would inhibit substrate binding. And, if the histidine residues were mutated independently, the mutation that dramatically affected the catalytic ability of the enzyme would show that the mutation was located in the active site of the enzyme. In order to experimentally test this hypothesis, two mutations were created: H188N and H129N. The two mutations were created by mutating a histidine at each of the sites into an asparagine. In order to quantify the effect of the mutations, the specific activity, average velocity, and average protein concentration of the reactions were measured and compared to wild-type EfumC. The results of the experiment showed that the H129N mutation had little effect on the enzymatic activity of the enzyme, as the specific activity of the enzyme was comparable to the wild-type enzyme. In contrast, the mutation dramatically reduced the specific activity of the catalytic reaction. These data strongly suggested that the H188 residue had a direct role in the catalytic mechanism of the enzyme and, therefore, that the H188 residue was located in the active site of the enzyme. Furthermore, it was concluded that the A-site was in fact the active site of the enzyme[2].
Structure and Function of the Fumurase Active Site
The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits and is located in a relatively deep pit that is removed from bulk solvent. The multi-subunit active site is comprised of atoms from residues 312-334 from subunit A, residues 182-200 from subunit C, and residues 129-145 from subunit D[3]. The residues that form the active site are N141b, T100b, S98b, E331c, K324c, N326c, His 188C, and a water molecule, W-26. It is speculated that the H188 and W-26 are two of the most vital active site residues. Furthermore, H188 and W-26 form a short hydrogen bond, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of . The cationic charge on W-26 plays an essential role in the stabilization of the double negative charge that is present on the aci-carboxylate at C4. Complex hydrogen bonding patterns in the active site involving T187, N141, H188, N362, and K324 also help stabilize the aci-carboxylate intermediate[2]. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.
