Fumarase is an enzyme that catalyzes the hydration and dehydration reaction between L-malate and fumarate. Fumarase C from E. coli (EfumC) is a Class II fumarase, which are iron-independent and themal-stable. Fumarases from eukaryotic organisms has been studied and characterized to a much greater extent compared to fumarase from E. coli. In an attempt to further characterize EfumC, crystallographic studies were conducted using the enzyme, however the studies produced some unexpected results. Crystallographic studies that utilized inhibitors related to the enzyme’s normal substrate found that the inhibitors pyromellitic acid and -trimethysilyl maleate bound to two different sites, which were named the A-site and B-site, respectfully. These contrasting results raised the question of which of the two sites was the active site?
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 – H188 in the A-site and H129 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. 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 H188N mutation dramatically reduced the specific activity of the catalytic reaction. These data provided strong evidence that H188 was located in the active site of the enzyme, therefore making the A-site the active site for the fumarase enzyme.
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