Sandbox Wabash 22 Fumarase
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Mutations of Fumarase--Mazin Hakim
Fumarase C is an important enzyme that catalyzes the hydration and dehydration equilibrium between L-malate and fumarate. Fumarase C from E.coli is highly homologous with the same enzyme from eukaryotes, and therefore is a prime analyte for the study of its reaction mechanism. In the study, "Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site"[1] conducted by Weaver et. al., there are various points of interest to address. these include the structure of the fumarase active site, what amino acids are present there, and free energy of the intermediate, but also extend to a discussion about the two possible locations of the active site and how this true active side was determined via a mutations and collection of data.
Possible Locations of the Active Site
The structure of the active site that is thought to consist of the overall catalytic process are the two basic groups, one responsible for deprotinating L-malate (B1) and another responsible for the removal of -OH from L-malate to form a water. Upon further crystallographic studies, there were two different active sites found, one that was a triple subunit binding site, the A site, of inhibitors citrate and pyromellitic acid and another single subunit labeled the B site.
There were many reasons the A site was thought to be the real active site. To start, no monomeric form of fumarase has ever been recorded as active, leaving the triple subunit A site the only active option. Also, since citrate, which is known to competitively inhibit the A site of the fumarase, was used in high concentration and the enzyme was measured as inactive. At site A, the wild type crystal structure was analyzed and the active site A was determined as relatively deep well, labeled in the following scene of the fumarase dimer using the residue . The B site of fumarase, located by the residue , is located more toward the surface of the monomer.
To determine which site of fumarase was the active site, the H188 and H129 were altered. If the A site was the active site, a change in catalytic activity would be apparent after mutating the H188 residue. If the B site was the active site, mutating the H129 residue would cause a large change in catalytic activity.
Data collected on the and H129N mutants, in which each histidine of the active site was replaced by a asparagine, was collected and the change in activities was observed in comparison to the activities of the wild types. The significance of the data is that there was no significant change in the mutation of H129 to H129N. The specific activities (u/mg) for the wild type and the mutant H129N were 116.2 +/- 14.0 and 143.7 +/- 10.0. As compared to the H188 and H188N, with activities of 116.2 +/- 14.0 and 0.55 +/- 0.044 u/mg respectively, the mutation of H188 was far more significant, demonstrating site A as the active site.
Structure and Role of the Active Site Residues
With the A site determined as the active site, the structure and important amino acids of the active site can be determined. The H188 has two proposed roles based on observations from X ray structure of H188N. The first one is the stabilization of the aci-carboxylate intermediate [2] through Coulombic (charge) interactions. The second role is the activation W26 to deprotinate C3 on malate.
The C4 carboxylate on L-malate contains the double negative charge of the transition state which is "coordinated through a series of hydrogen bonds from T187-OG1 and N141-ND2 to the O3 oxygen atom and from H188-NE2, N326-ND2, and K324-NZ to the O4 atom."[3] The second function for H188 is the W-26 activation which helps out with the deprotination of C3 on L-malate. Lastly, shown via the represented by E331 and H188 in the quaternary structure is an activator of water which plays a vital role in removing a the C3 protons from L-malate.
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