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, 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 is thought to involve 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 [1] mutated the Histidine side chain, and created two fumarase mutants H129N and H188N. These mutants were developed to hinder the catalytic activity of fumarase. Data was gathered from crystal structure analyses, and activity measurements to confirm the active site [1].
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