Sandbox Wabash 06 Fumarase

Debate

of fumarase C from E. coli have been made using PCR and recombinant DNA.  Two different carboxylic acid binding sites (A+B) were observed in the crystal structures of the WT inhibited forms of the enzyme.  The has L-malate bound at active site A.    

Crystallographic studies with several inhibitors including pyromellitic acid and B-trimethylsilyl maleate yielded interesting results. While both inhibitors are related to the normal substrate, each was found bound at different sites. The binding site of inhibitors citrate and pyromellitic acid was deemed the A site while a second site containing L-malate and B-trimethylsilyl maleate was labeled as the B-site. The first argument for site A being the active site was that the A site was formed by 3 of the 4 subunits. Secondly, citrate was used at high concentrations as a crystallizing agent and is known to competitively inhibit fumarase. As such, in X-ray studies, citrate was not able to be removed readily from the specimen preparation and it pointed to the A site. In regards to the B site, it was first noted that atoms of a single subunit formed the B site. Strong arguments were then made against the B site as no active monomeric form of fumarase has ever been described. ^[1]

True Active Site

In lieu of the fact that a histidine side chain was one of the bases in the catalytic reaction, in order to determine which of the two sites was indeed the active site, histidines could be observed. By mutating the histidines at the two active sites, one would be able to determine that if the A-site was active, changing the would dramatically affect the catalytic activity. Rather, if the B-site was the active site, then a mutation in would affect catalysis. ^[2]

Structural highlights

A histidine at each of the sites was mutated to an asparagine. The H188N mutation at the A-site resulted in a large decrease in specific activity. Rather, the H129N mutation at the B-site resulted in no change in activity. ^[3]

This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.