Sandbox Wabash 02 Fumarase

Determination of the True Active Site of Fumarase C from E. Coli.

Fumarase C from E. coli is an enzyme homologous with the cytosolic and mitochondrial enzymes found in eukaryotic cells. It catalyzes the hydration/dehydration reaction between the metabolites L-malate and fumarate. Eukaryotic fumarases have been studied extensively by classical kinetic and isotope labeling methods (Hill & Teipel, 1971; Cleveland,1977). Fumarase from E. coli is less well characterized. Two basic groups are thought to be involved in the overall catalytic process. The first (B1) is responsible for the removal of a proton from the C3 position of L-malate, resulting in a carbanion stabilized by an acid-carboxylate intermediate formed at C4. The last stage of the catalytic process is thought to involve a second basic group on the protein labeled B2. In the direction of fumarate production, this basic group would be protonated and the removal of the -OH from C2 results in the formation of a water molecule.The proton at B1 in Figure 1 has unusual properties and is believed to be removed as the next substrate molecule binds (Rose et al., 1992). The crystallographic studies of wild-type fumarase indicated that the enzyme had an unusual subunit arrangement with a core of 20 alpha-helices, 5 from each of the subunits (Weaver et al., 1995). Subsequent crystallographic studies with several inhibitors including pyromellitic acid and beta-trimethylsilyl maleate produced some unexpected results. Although both are related to the normal substrate, they are bound at different sites. The original tungstate site, a heavy atom derivative, was also the binding site of the inhibitors citrate and pyromellitic acid. This was labeled the A-site and is comprised of atoms from three of the four subunits (Weaver & Banaszak, 1996). The second site contained L-malate in the native crystals and beta-trimethylsilyl maleate in another crystallographic study (Weaver & Banaszak, 1996), and was labeled the B-site and was formed by atoms from a single subunit. The results led to the dilemma as to which of the two sites was the active site.

Structure of the Active Site of Fumarase. You may include any references to papers as in: the use of JSmol in Proteopedia ^[1] or to the article describing Jmol ^[2] to the rescue.

Function

Disease

Relevance

Structural highlights

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