Sandbox Wabash 28 Fumarase

Fumarase's debated active site

is a highly conserved enzyme found in bacteria as well as mitochondria of eukaryotes which catalyzes the hydration/dehydration of fumarate and L-malate, respectively. The protein, which consists of two dimers laden with α-helices, initially crystalized as a dimer making conclusions about its active site problematic. However, subsequent crystalizations with various inhibitors showed two potential active sites - the locations at which the inhibitors bound: the first discovered theoretical active site is located deep in the interior of the tetramer and was inhibited by pyromelletic acid and citrate - the latter of which is found in the potential active site; β-trimethylsilyl maleate inhibition resulted in the discover of found at the exterior of each subunit. In either case, a basic His residue is believed to be the origin of catalytic function and two such residues were found (His 188 in site A and His 129 in site B). To conclusively determine whether A or B is the active site, site-directed mutagenesis in E. coli was conducted.

Theoretical mechanism

L-malate is believed to be dehydrated by fumarase into fumarate using basic His residues in its active site.

Fumarase mechanism

One of the His deprotonates the γ-C producing an anion stabilized by the aci-carboxylate carbanion intermediate. The hydrogenation of the alcohol by a second, previously protonated His base renders the alcohol a good leaving group. The closing of the aci-carboxylate into a carboxylate and alkene by pushing off the water produces fumarate.

The mechanism can also work in reverse as it is the aci-carboxylate intermediate that is believed to be most stabilized by fumarase. In this direction, a water molecule attacks the alkene to produce the aci-carboxylate carbanion. This high energy structure quickly deprotonates a local, protonated His to produce L-malate. In both cases, H-bonds to the carboxylate groups hold the substrate in place. The alternation of protonation and deprotonation for the His residues locally stabilizes the charged intermediates: the negatively charaged aci-carboxylate anion is next to a positively charged, protonated His residue. Ultimately, fumarase catalyzes the reaction by making available the acid/base catalysis required for these reactions in water.

Determination of active site

As stated earlier and suggested by the theorized mechanism, the use of His residues which are readily protonated and deprotonated at physiological pHs and the binding of inhibitors to two different sites created two targets: His 188 for site A and His 129 for site B. To determine which was the true active site, single-site directed mutagenesis of His to Asn was conducted. The activity of the resultant fumarase mutants (H188N and H129N) were compared against the wild type to determine changes in catalytic ability. Crystolography was performed as well to observe changes in the protein structure (bond angles, distances between atoms, orientation of substrate, etc.). The choice of Asn limited changes in steric bulk as well as folding from different H-bonding interactions (both His and Asn residues act as H-bond acceptors and donors).

(H188N) produced an enzyme with approximately 200-fold less specific activity than the wild type. Crystalographic data showed little to no changes in site B whereas the presence of N188 encouraged increased H-bonding with a water molecule instead of the ligand (represented by the inhibitor citrate in the crystal).

Although modifications at (H129N) also changed H-bonding patterns in the site. The net result was little to no conformation change at either site A or B. The catalytic studies demonstrated a small, but significantly larger specific activity in the mutant. Citrate (the inhibitor representative of the substrate) remained bound to as with the wild type.

Cohesively, these results are indicative that site A (especially H188) is the catalytic active site.

References