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Sandbox Wabash 13
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(Difference between revisions)
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== Early difficulties distinguishing between two adjacent dicarboxylic acid binding sites in fumarase C == | == Early difficulties distinguishing between two adjacent dicarboxylic acid binding sites in fumarase C == | ||
| - | In early studies of <scene name='72/726365/Fumarase_quaternary_structure/1'>fumarase C</scene> 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 <scene name='72/726365/Active_site_histidines/1'>histidine residue</scene> 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 H188 (located in the A- site) and H129 (located in the B-site) 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. | + | In early studies of <scene name='72/726365/Fumarase_quaternary_structure/1'>fumarase C</scene> 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 <scene name='72/726365/Active_site_histidines/1'>histidine residue</scene> 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 H188 (located in the A- site) and H129 (located in the B-site) 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<ref>PMID:9098893</ref>. |
== Data == | == 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: | + | 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<ref>PMID:9098893</ref>. The group further investigated the fumarase mutations to determine the structural implications and observed the following: |
''Active Site A:'' | ''Active Site A:'' | ||
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== References == | == References == | ||
| - | < | + | <references/> |
Revision as of 19:01, 28 February 2016
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