Sandbox Wabash 02 Fumarase
From Proteopedia
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Determination of the True Active Site of Fumarase C from E. Coli
Fumarase C from E. coli is an enzyme homologous to the cytosolic and mitochondrial enzymes found in eukaryotic cells. It catalyzes the hydration/dehydration reaction between the metabolites L-malate and fumarate. Fumarase C is a tetrameric enzyme composed of four identical sub-units of 50 kDa each while each subunit is mostly composed of α- helices. Previous studies have found that fumarase has two different carboxylic acid binding sites ( and ) in both wild-type and mutated forms of the enzyme. Results from these studies led to the dilemma as to which of the two sites was the active site. Because the A site is deeper at the center of fumarase and involves residues from three of the four subunits, it received initial support to be the true active site. Further experiments were done to determine which of these binding sites was the true active site. The H129N and H188N mutants were generated to resolve the two site problem. If the A-site was the active site, changing H188 should dramatically affect the catalytic activity. Conversely, if the B-site was the active site then a mutation at H129 should affect catalysis. A histidine to asparagine mutation at the A site resulted in a large decrease in specific activity, whereas a histidine to asparagine mutation at the B site resulted in essentially no effect. Therefore, it was determined that the A site is the active site, although the B site binds to substrates and their analogs as well with a lower ligand binding affinity.[1]
Structure of the Active Site of Fumarase
Eukaryotic fumarase catalysis kinetics, stereochemical characteristics and mutant kinetics have been extensively studied. The determined active A site contains residues of Asn 141, Thr 100, Glu 331, Ser 98, His 188, Asn 326 and Lys 324. Asn 141 is especially important because it is one of the amino acids that forms hydrogen bonds with water in the active site. Water and this residue are vital to the enzyme’s mechanism and function. Site A is in a relatively deep pit removed from bulk solvent and contained a bound water. One of the side chains interacting with this water molecule is H188. In the crystallographic coordinates of the wild-type enzyme, the water molecule forms a short hydrogen bond, 2.5 A, with the imidazole ring of H188. The side chain of H188 is also within hydrogen bonding distance to an oxygen atom of bound citrate or pyromellitic acid. The B-site is closer to the surface of the enzyme. There are three principal interactions between the ligand and wild-type fumarase at the B-site, and the A- and B-sites are linked by residues 131 to 140 in a single subunit. Main chain hydrogen bonds between the oxygen atoms of the bound ligand and main chain -NHs of D132 and N131 on the N-terminal end of the pi-helix are important to stabilization at the B-site. Oxygen atoms of the other carboxylate of the ligand at the B-site are hydrogen bonded to R126-NE and H129-NDI. The hydrogen bonds between side chain atoms of N135 and N103, and between N103 and S140 form an indirect connection between the B- and the A-site. H129 is the only basic group close to a ligand bound at the B-site.[2]
References
- ↑ 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
- ↑ 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
