is an enzyme that catalyzes the reactions of the hydration and dehydration of fumarate to malate, and vice versa(). When the enzyme was first examined, there was a debate over the location of the active site of the enzyme. This confusion was due to the fact that there were two carboxylic acid binding sites on the fumarase that had the potential to be the active site. These binding sites were both present in wild type and inhibited forms of the enzyme. In order to determine which carboxylic acid binding site was the actual active site, mutants of these two binding sites were observed. The first site was designated "A". This site is composed of several different subunits, primarily , Serine 98, Threonine 100, and Asparagine 41. The second site was designated "B". This site was primarily composed of , Asparagine 131, Aspartic Acid 132, and Arginine 126. Since both sites had a histidine that was integral to the binding site, the mutation that was conducted changed the histidines in each binding site. This allowed more control to be had over the mutations because the variable of different mutated amino acids doing different things to the fumarase was now controlled. The experiment was conducted using E. coli and then performing PCR using recombinant DNA to insert and multiply the mutated enzyme. The when it was mutated, resulted in a significant decrease in activity of the enzyme. The had no significant effect upon the activity of the fumarase. This indicates that the active site of fumarase is actually "A" because that is the binding site that the activity was affected by.
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