Sandbox Wabash 20 Fumarase

Fumarase C is the stable fumarase found in E. coli acting as a "non-iron-containing enzyme" found in eukaryotic cells. Fumarase is a vital enzyme in eukaryotic cells because it has the ability to catalyze dehydration and hydration reactions between L-malate and fumarate,^[1] a reaction found in the Krebs Cycle. This leaves a particular importance on the active site of fumarase, which dictates the catalytic activity of the enzyme. The first stage of the of the catalytic process is the removal of a proton from the C3 position of L-malate followed by the a deprotanation and removal of an –OH group from C2 resulting in a water molecule being formed. The presence or lack thereof of these processes is a key indicator for catalytic activity and will be used in determining the true active site of Fumarase C.

Active Site Debate

While fumarases have been historically well researched, the active site of Fumarase C has been a point of contention. The candidates for the potential actives sites are two carboxylic acid binding sites named A-site and B-site. To determine the true active site, the histidine at each site was mutated into an asparagine because the recombinant form of the protein includes a “histidine arm on the C-terminal”. The H188N mutation is associated with the A-site and the H129N mutation is associated with the B-site.

Mutations for both the A-site and B-site were created using PCR and recombinant DNA. The protocol by Weaver et al. notes that 1 μg of pASK40/fumarase vector was transformed into 100 μL of DH5α cells. Specific primers were used to create the correct fragments and were then separated using 1% agarose gel electrophoresis and subsequently extracted. The cells were then grown and purified using nickel column chromatography and SDS-PAGE.

In order to test the hypothesis, the specific activity of the wild type and mutants was measured, as shown in the table below. As the table notes, the mutation in H129N, which correlates to B-site, had relatively little change to the wild type. On the other hand, a mutation in H188N which corresponds to A-site, dramatically reduced the activity, almost to zero. Because of this dramatic reduction, these specificity data support the notion that the A-site is the true active site of Fumarase C.

X-ray crystal structure was also used to observe the effects of various mutations local conformation.

Structure of Active Site

References

1. ↑ Weaver T, Lees M, Banaszak L. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site. Protein Sci. 1997 Apr;6(4):834-42. PMID:9098893