Sandbox Wabash 28 Fumarase
From Proteopedia
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 residues in its active site.
One of the bases deprotonates the γ-C producing an anion stabilized by the aci-carboxylate carbanion intermediate. The hydrogenation of the alcohol by a second, previously protonated 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 base to produce L-malate.
