Fumarase Binding Sites
Fumarase is a common enzyme in the metabolization of amino acids. Two Carboxylic acid binding sites have been observed in wild-type fumarase, resulting in a debate over which is the active site. In a 1997 research article titled: Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site", some intriguing evidence regarding the aforementioned binding sights was presented. These findings were made unconventionally through the use of fumarase, originating from E. Coli, that was mutated. The mutation process was carried out by enlisting Polymerase Chain Reactions (PCR). PCR is effective due to its ability to produce sufficient amounts of products that have reacted at both binding sites. The mutations were reportedly successful by replacing existing histidines with asparagines at both binding sites. The article denotes the binding sites A and B. H188N is the mutation at site A as H192N is for site B. This research reported that site A mutation displayed a considerable decrease in specific activity and that site B mutation resulted in nothing considerable, to which the research concluded that site A is the actual active site. B, therefore, is considered the allosteric site of fumarase despite its similarities.
Mechanistic Process
In short, fumarase possesses a water molecule at its active site that catalyzes the hydrolyzation reaction of fumarate to produce L-malate. However, this reaction is reversible. The research article hypothesizes that the fumarase catalyzed conversion of L-malate to fumarate proceeds in the following fashion:
1. Deprotonation of C3 position carbon of L-malate
2. Formation of carbanion stabilized by aci-carboxylate intermediate at C4
3. protonation of hydroxyl group on C2 position Carbon
4. Water leaving group
