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.
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