Fumarase

Overview

Fumarase, also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.

Stucture: will the real active site please stand?

Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. ^[1]. But these changes could account for regulation...so which site is the true active site?

In order to answer this question, an experiment that tested each of the sites independently was conducted. Both sites contain histidine residues: in the A-site and in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. The results of the experiment showed that the H129N mutation had little effect on the enzymatic activity of the enzyme, as the specific activity of the enzyme was comparable to the wild-type enzyme. In contrast, the mutation dramatically reduced the specific activity of the catalytic reaction. These data strongly suggested that the H188 residue had a direct role in the catalytic mechanism of the enzyme and, therefore, that the H188 residue was located in the active site of the enzyme. This lead to the conclusion that that the A-site was in fact the active site of the enzyme^[2].

Active Site Characteristics

The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in ) and is located in a relatively deep pit that is removed from bulk solvent ^[3]. The residues that form the include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the is the most important active site residue, activating the water through a , which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate^[2]. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.

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References

1. ↑ Weaver,T. Structure of free fumarase C from Escherichia coli. Acta Crystallographica (2005), D61, 1395-1401. [http://dx.doi.org/10.1107/S0907444905024194 doi:10.1107/S0907444905024194]
2. ↑ ^{2.0 2.1} Weaver T, Lees M, Banaszak L. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site. Protein Sci. 1997 Apr;6(4):834-42. PMID:9098893
3. ↑ Weaver TM, Levitt DG, Donnelly MI, Stevens PP, Banaszak LJ. The multisubunit active site of fumarase C from Escherichia coli. Nat Struct Biol. 1995 Aug;2(8):654-62. PMID:7552727