2fus
From Proteopedia
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==Overview== | ==Overview== | ||
| - | Two mutant forms of fumarase C from E. coli have been made using PCR and | + | Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Fumarate hydratase]] | [[Category: Fumarate hydratase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Banaszak, L | + | [[Category: Banaszak, L J.]] |
[[Category: Lees, M.]] | [[Category: Lees, M.]] | ||
| - | [[Category: Weaver, T | + | [[Category: Weaver, T M.]] |
[[Category: CIT]] | [[Category: CIT]] | ||
[[Category: carbon oxygen lyase]] | [[Category: carbon oxygen lyase]] | ||
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[[Category: kreb's cycle enzyme]] | [[Category: kreb's cycle enzyme]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:25:19 2008'' |
Revision as of 15:25, 21 February 2008
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MUTATIONS OF FUMARASE THAT DISTINGUISH BETWEEN THE ACTIVE SITE AND A NEARBY DICARBOXYLIC ACID BINDING SITE
Overview
Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.
About this Structure
2FUS is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Fumarate hydratase, with EC number 4.2.1.2 Known structural/functional Site: . Full crystallographic information is available from OCA.
Reference
Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site., Weaver T, Lees M, Banaszak L, Protein Sci. 1997 Apr;6(4):834-42. PMID:9098893
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