Sandbox Wabash 10 Fumarase

Fumarase C is an enzyme from E. coli (EFumC) that catalyzes the hydration/dehydration reaction between L-malate and fumarate. It catalyzes the hydration of the double bond to form malate. The hydration reaction continues through a carbanion transition state. It has no known metal ion requirement and has a high degree of homology with eukaryotic enzymes. Its homology with cytosolic and mitochondrial enzymes in eukaryotic cells makes it ideal for research. Through x-ray crystallography it has been shown that the enzyme is comprised of a unusual subunit arrangement composed of a core of 20 α-helices, 5 in each of the subunits (shown here is ), and it is a tetrameric enzyme with each monomer containing approximately 460 residues.

The Debated Fumarase C Active Site

The overall catalytic mechanism of Fumarase drives fumarate formation from L-malate. A water molecule is removed from L-malate to generate fumarate. The first step of this is through a proton removal, and followed by OH- ion removal. The debate for the active site of fumarase involves two active sites that both contain carboxylic acid binding sites; the A and B site. Biochemical data suggests that the Histidine side chain is one of the bases participating in the catalytic reaction ^[1]. In order to determine which is the actual active site of fumarase, Weaver mutated the Histidine side chain, and created two fumarase mutants H129N and H188N^[1]. The represents the location of the targeted mutation within site A, and represents the location of the mutated residue in site B. The A-site appears to be in a relatively deep pit, and the B-site appears to be nearer to the surface edge of the active site pit. The B-site is formed from a single subunit of the tetramer and includes atoms from residue R126, H129, N131, and D132 ^[1]. H129 was mutated because it is the only potential side chain that could serve as a catalytic base in the B-site. Each of these residues (H188 & H129) were replaced by asparagine to determine the role of the A and B sites in the enzymatic catalysis of L-malate, in order to hinder the catalytic activity of fumarase. Data was gathered from crystal structure analyses, and activity measurements to confirm the actual active site ^[1]. Through a nickel agarose column and subsequent SDS-PAGE, they purified the histidine tagged protein ^[1]. Subsequently, they calculated the specific activities of the wild-type fumarase and the histidine mutants H129N and H188N. Weaver observed that the H188N mutant drastically affected the catalytic reaction, showing an avg activity of 9.62 μ/mL, as opposed to the wild type with 4920.0 μ/mL and the H129N mutant with 2080 μ/mL ^[1].These results support his hypothesis that site A was in fact the active site, by changing the H188 residue, they dramatically affected the catalytic activity of the enzyme ^[1]. Additionally, this was further supported by eliminating the HIS188 from fumarase, in the absence of HIS188 it effectively reduced the binding of citrate. They also note that the A-site (active site) in the structure of H129N, was unchanged by a mutation at H129 residue further showing that it is HIS188 that is necessary for the catalytic mechanism of the active site within fumarase ^[1].

Actual Active Site of Fumarase

, shown through studying the wild type conformation of Fumarase. It has four protein ligands from two subunits including residues T100b, S98B, N141b, H188c, E331c, and an active site water molecule (W-26) ^[1]. The residues from three of the chains form the active site (site A) of the enzyme. In its native conformation the active site water W-26 is bonded to five different atoms, including a bound citrate ion.This is also supported by the fact that in the H129N structure, W-26 acts as a donor and the acceptor atoms: H188-NE2, N141-OD1, S98-OG, T100-OG, these are the same four protein atoms reported in the wild type crystal structure from the bound citrate ^[1]. In this orientation the O4 atom no longer maintains hydrogen bonding distance but is positioned 3.67A from W-26 ^[1].