Sandbox Wabash 13

Distinguishing between the active site and a nearby dicarboxylic acid binding site in Fumarase C

Background

In early studies of in "E. Coli", two carboxylic acid binding sites, subsequently named A- and B-, were observed in the wild type crystal structure. The structures of these two sites was 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 but it could not be verified without first mutating both active sites in order to determine the activity of the reactions. Prior data had suggested that a was the critical base in the catalysis and therefore H188 (located in the A- site) and H129 (located in the B-site) were mutated to asparagines in the hopes that this residue mutagenesis would prompt a dramatic catalytic affect in the active site and minimal affect in the other carboxylic acid binding site.

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. 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). H188N: Only residues N141b, S98b and N188d maintain interactions with W-26. T100b and citrate 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 site. H129N: Side chains N131, D132 and N129 hydrogen bond to each other and prevent interactions with R126 and the ligand alike. H188N: No change observed from WT (All 5 interactions were maintained with the ligand).

Relevance

Structural highlights

References

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