Sandbox Wabash 10 Fumarase
From Proteopedia
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=='''The Debated Fumarase C Active Site'''== | =='''The Debated Fumarase C Active Site'''== | ||
| - | The overall catalytic mechanism of Fumarase drives fumarate formation from L-malate. A water molecule is removed from L-malate to generate fumarate. The first step of this is through a proton removal, and followed by OH- ion removal. The debate for the active site of fumarase involves two active sites that both contain carboxylic acid binding sites; the A and B site. Biochemical data suggests that the Histidine side chain is one of the bases participating in the catalytic reaction <ref name= "Weaver">PMID:9098893</ref> In order to determine which is the actual active site of fumarase, Weaver <ref name= "Weaver">PMID:9098893</ref> mutated the Histidine side chain, and created two fumarase mutants H129N and H188N. These mutants were developed to hinder the catalytic activity of fumarase. Data was gathered from crystal structure analyses, and activity measurements to confirm the active site <ref name= "Weaver">PMID:9098893</ref>. Through a nickel agarose column he purified the histidine tagged proteins | + | The overall catalytic mechanism of Fumarase drives fumarate formation from L-malate. A water molecule is removed from L-malate to generate fumarate. The first step of this is through a proton removal, and followed by OH- ion removal. The debate for the active site of fumarase involves two active sites that both contain carboxylic acid binding sites; the A and B site. Biochemical data suggests that the Histidine side chain is one of the bases participating in the catalytic reaction <ref name= "Weaver">PMID:9098893</ref> In order to determine which is the actual active site of fumarase, Weaver <ref name= "Weaver">PMID:9098893</ref> mutated the Histidine side chain, and created two fumarase mutants H129N and H188N. These mutants were developed to hinder the catalytic activity of fumarase. Data was gathered from crystal structure analyses, and activity measurements to confirm the active site <ref name= "Weaver">PMID:9098893</ref>. Through a nickel agarose column he purified the histidine tagged proteins<ref name= "Weaver">PMID:9098893</ref>. Subsequently, he calculated the specific activities of the wild-type fumarase and the histidine mutants H129N and H188N. Weaver observed that the H188N mutation drastically affected the catalytic reaction, showing an avg activity of 9.62 μ/mL as opposed to the wild type with 4920.0 μ/mL and the H129N mutant with 2080 μ/mL <ref name= "Weaver">PMID:9098893</ref>. |
Revision as of 02:49, 29 February 2016
Fumarase
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References
- ↑ 1.0 1.1 1.2 1.3 1.4 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[1]
