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, has four protein ligands from two subunits including residues T100b, S98B, N141b, H188c, E331c, and an active site water molecule (W-26) ^[1]. 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].