Background
Fumarase catalyzes the reversible hydration/dehydration reaction between L-malate and fumarate through acid/base catalysis. The removal of an H+ from the C-3 position of L-malate by a basic group (Histidine) of the enzyme leads to a carbanion intermediate (aci-carboxylate). The -OH group attached to the C-2 position of the intermediate then leaves as an OH- ion through a protonated basic group that leads to the formation of H20.
Early difficulties distinguishing between two adjacent dicarboxylic acid binding sites in fumarase C
In early studies of in E. Coli, two adjacent carboxylic acid binding sites, subsequently named A- and B-, were observed in the wild type crystal structure. The location of these two anion sites led to difficulty in identifying the proper active site of the enzyme. The structures were considerably different as site A contained atoms of three of the four subunits of fumarase C whereas site B contained only atoms from a single subunit of the tetramer. However, it was heavily believed that the carboxylic acid binding site A- was the location of the fumarase active site as previous studies had not described a fumarase as active in a monomeric form. Prior data had also suggested that a histidine was the critical base in the catalysis. Verification could not be obtained without first mutating both active sites in order to determine the activity of the reactions and therefore and were mutated to asparagine with the understanding that this residue mutagenesis would prompt a dramatic catalytic affect in the active site and minimal affect in the other carboxylic acid binding site[1].
Data
Weaver et. al., discovered that upon mutagenesis of the H129 residue to H129N, the specific activity increased slightly from 116.1 (u/mg)^a for the WT fumarase to 143.7 (u/mg)^a, indicating that site B was indeed not the active site. Upon mutagenesis of the H188 residue to H188N, the specific activity decreased by slightly over 211 times less than that of the WT to 0.55 (u/mg)^a, assuring that location A was the active site[2]. The group further investigated the fumarase mutations to determine the structural implications and observed the following:
Active Site A:
WT: Side chains N141b, T100b, S98b, H188d and the inhibitor chosen (citrate) are hydrogen bonded a water molecule (W-26) located in the active site.
H129N: No change observed from WT (all 5 interactions are maintained with W-26).
: Only residues N141b, S98b and N188d maintain interactions with W-26. T100b and citrate (malate in the pictured scene) are unable to hydrogen bond to W-26 due to the mutagenesis.
Active Site B:
WT: Side chains N131, D132, H129 and R126 form a total of 5 hydrogen bonding interactions with the ligand in the active site.
H129N: Side chains N131, D132 and N129 hydrogen bond to each other and prevent interactions with R126 and the ligand alike.
: No change observed from WT (All 5 interactions were maintained with the ligand).
Conclusion
In the presence of the mutation , L-Malate is unable to bind to the active site A but can still bind with H129 (site B), detailing the initial confusion in determining the correct active site location. Combined with the specific data activity gathered from kinetic analysis after mutagenesis, it could be confirmed that the histidine at site A (H188) was crucial to the conversion of L-malate to fumarate via base catalysis and therefore site A was the active site.
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
