Sandbox Wabash

Overview

Fumarase C is an enzyme found in eukaryotic cells that catalyzes both hydration and dehydration reactions between L-malate and fumarate. Both of these compounds are intermediates within the citric acid cycle, one of the major cellular respiratory pathways that oxidizes acetyl CoA from sugars. Fumarase C is a tetrameric protein, and each monomer possessing approximately 460 amino acids.

Active Site Debate

Previous studies using fumarase C inhibitors pyromellitic acid and β-trimethylsilyl maleate discovered that these compounds inhibited fumarase C activity with at two different sites: pyromellitic acid bound to the designated “A-site”, which is composed by three of the four fumarase C subunits, whereas β-trimethylsilyl bound to the “B-site”, composed of a single subunit. Todd Weaver and Leonard Banaszak (1996) investigated both the A-site and B-site in fumarase C to understand their functions more clearly. The A-site is in a relatively deep pit in fumarase C and notably contains the residue , which interacts with a bound water molecule that is utilized to catalyze the reactions. The B-site, much closer to the surface of the enzyme, also possesses a His-residue (). Previous studies implied that a histidine residue was a major proponent in fumarase C activity, so Weaver, Mason Lees, and Banaszak (1997) developed mutations at the A site () and B site (). After calculating the specific activities for the wild type and mutated forms of fumarase C, they found that though the wild type and H129 had similar levels of activity, the H188N mutant yielded significantly lower levels of activity. In addition, crystallography data of the H188N and H129N mutants revealed that the structural conformation of H129N varied little in comparison to the wild type form. Unlike H129N or the wild type fumarase C, the H188N mutant was unable to bind to citrate due to a major change in active site conformation, which would limit its role in the citric acid cycle. Combined, these two pieces of evidence suggest that the A-site in fumarase C is indeed the active site.

Function

The (i.e. A-site) is composed of the residues His-188, Thr-100, Ser-98, and Asn-141. His-188 non-covalently interacts with a water molecule, which is used to stabilize the hydration/dehydration reaction intermediates of fumarase C by removing a proton from the C3 position of the substrate. In addition, His-188 directly binds to the C4 carboxylate group, mediating hydrogen-bond interactions of the substrate with other amino acid residues.

References

1. Weaver T, Lees M, Banaszak L. 1997. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site. Protein Science 6: 834-842.