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”^[2]. The mutation is associated with the A-site and the 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^[3].

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^[4] 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

As noted above, the A-site is believed to be the active site for Fumrase. Histidine (H188) is the most important of the amino acids in the active site as seen by the reduction of activity after it’s transformation into asparagine. Residues 131 to 140 are linked to the active site. Weaver notes, “main chain hydrogen bonds between the oxygen atoms of the bound ligand and the main chain –NHs of D132 and N131 on the N-terminus of the π –helix are important to the stabilization of the B-site”^[5].

While the A-site is the true active site, there is a “dual role” for the H188 in the site. After the mutation in the histidine, the replaced asparagine side chain still interacts with water, although slightly moved. The shift is approximately 0.70 A. Another observation with the H188 mutation is that the absence of the histidine reduces binding of citrate. Furthermore, the A-site is composed of atoms from of R126, H129, N131, and D132 to create most of the H-bonding partners. This is important because L-malate contains a double negative charge in the aci-carboxylate intermediate. In the first step of the fumerase reaction mechanism, the removal of a proton from the C3 position of L-malate results in a carbanion which is stabilized by the aci-carboxylate intermediate. This series of hydrogen bonding is able to properly position the C3 and C4 atoms of L-malate. H188 ultimately increases the basicity of the active site water molecule allowing a positive charge to stabilize the double negative charge of the C4 intermediate.

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
2. ↑ 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
3. ↑ 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
4. ↑ 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
5. ↑ 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

Cystic Fibrosis Homework

Mutations associated with cystic fibrosis can have either a class defect in conduction or regulation. Amino acids associated with are R117H, R334W, R347P. Overall, these mutations are located in the inner portions of the molecule. This area is vital in the actual passage of chloride ions which the transmembrane regulator facilitates. A mutation in these locations could block the flow of chloride ion or prevent the substance from efficiently moving. A second type of mutation is a mutation, which is a class III mutation where the protein is made and is positioned properly, but does not function properly. Amino acids associated with regulation mutations are G551D, G551S, G1224E, G1255P, and G1349D mutations. These mutations are generally located on the outer portions of the membrane. Epithelial chloride flux is known to be mediated by phosphorylation of the CFTR. Phosphorylation affects ATP binding (by ATP gated ion receptors located at the surface) which ultimately inhibits the channel from opening. A mutation in these amino acids may then prevent phosphorylation.