Sandbox wabash 07 Fumarase

Fumarase C from E. coli catalyzes the hydration/dehydration reaction between the L-malate and fumarate. It belongs to a group of enzymes which are tetrameric with each monomer containing about 460 amino acids. There are two basic groups believed to involve in the catalytic process.The first group is responsible for the removal of a proton from the . The resulting carbanion is stabilized by an aci-carboxylate intermediate formed at . The second basic group on the protein is thought to involve in the last stage of the catalytic process. When producing fumarate, the second basic group would be protonated and the removal of the -OH from C2 results in the formation of a water molecule. The proton in the first group has unusual properties and is believed to be removed as the next substrate molecule binds.This enzyme also has an unusual subunit arrangement as it has a core consisting 20 alpha helices, 5 from each of the subunits ^[1].

Two Possible Active Sites

The crystallographic studies using pyromellitic acid and beta-trimethylsilyl maleate indicated the presence of two possible binding sites. The original tungstate site, a heavy atom derivative, is one of the possible sites called . It's found to be a binding site of the inhibitors citrate and pyromellitic acid. This site is comprised of atoms from three of the four subunits and is relatively deep in the structure of the enzyme. However, another crystallographic study identified a second blinding site as it contained L-malate and beta-trimethylsilyl maleate as well. This second possible binding site called is formed by atoms from only one subunit and is relatively closer to the surface of the enzyme^[2]. Therefore there was a dilemma about which site is the true active site for fumarase C. Nonetheless, since fumarase is only active as a tetramer and only A-site contains atoms from three of the four subunits, it received the initial support for being the true active site.

Determination of True Active Site

Research suggested that a histidine side chain was one of the bases participating in the catalytic reaction therefore testing whether HIS 129 or HIS 188 affected catalytic activity offered a way of resolving the two site dilemma. If the was the active site, mutating HIS 188 should greatly change the catalytic activity.On the other hand, if the B-site was the active site then a mutation instead at HIS 129 should affect the catalytic process. To conduct the experiment, two mutant fumarases H129N and H188N were studied using crystal structure analysis and activity measurement. In both mutants, the histidine are mutated to asparagine. Two evidence supporting A-site is the true active site was provided by the experiments. First, the activity result data indicated that H129N (specific activity 143.7 ± 10.0 u/mg) have about the same specific activity as the wild type (specific activity 116.2 ± 14.0 u/mg) while the H188N mutation dramatically affects the catalytic reaction - it's specific activity (0.55 ± 0.044 u/mg) is about 200 times less than that of wild type or HI29N. The crystal structure analysis provided the second evidence. The crystal structure of H129N showed that the mutated protein had essentially the same conformation as the wild type but appeared to dramatically reduce binding of ligands at the B-site while the crystal structure of H188N showed that the absence of the histidine side chain effectively reduces binding of citrate so that it is missing in the electron density maps^[3].Therefore, in conclusion, the A-site is the actual active binding site.

Active Site Structure

The determined of fumarase involves ASN 141, THR 100, GLU 331, SER 98, HIS 188, ASN 326 and LYS 324. Amoung these residues, the most important one is HIS 188. In the fumarase active form, the imidazole ring of HIS 188 is oriented such that it forms a short hydrogen bond to a water molecule and GLU 331. GLU 331 may have contributed to increasing the basicity of HIS 188. This relay effect may in turn be passed to the active site water.It's worth noting that the current experimental data implies that HIS 188 does not interact directly with the , instead, it's the activated water molecule W-26 that removes the C3 proton of L-malate^[4]. HIS 188 has two functions in the catalytic process. First, it assists in binding the of the substrate through Coulombic interactions.The C4 carboxylate of L-malate is the location of the double negative charge of the aci-carboxylate intermediate. The second function of HIS 188 is to activate W-26 to facilitate proton removal from the C3 position. In this process, HIS 188 increases the basicity of the active site water molecule W-26. After C3 proton is moved to the water molecule, the extra Coulombic plus charge is close enough to aid in stabilization of the doubly negative charge present on the aci-carboxylate at C4^[5].