Sandbox Wabash 15 Fumarase

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==Crystallographic Determination of the Active Site in Fumarase C from E. coli==
==Crystallographic Determination of the Active Site in Fumarase C from E. coli==
'''Fumarase C''' is an enzyme from Escherichia coli that catalyzes the stereospecific, reversible interconversion between L-malate and fumarate via the hydrolysis of the carbon-oxygen bond. The hydration/dehydration reaction catalyzed by fumarase C is of particular interest, because it plays an essential role in the metabolic citric acid cycle<ref>PMID:8909293</ref>. Fumarases from eukaryotic organisms have been studied and characterized to a much greater extent, compared to fumarase C from E. coli. In an attempt to further characterize EfumC, crystallographic studies were conducted using the enzyme, however the studies produced some unexpected results. Crystal structures of EfumC revealed that the enzyme has two dicarboxylate binding sites. More specifically, crystallographic studies that utilized inhibitors related to the enzyme’s normal substrate found that the inhibitors pyromellitic acid and β-trimethysilyl maleate bound to two different sites, which were named the A-site and B-site, respectfully<ref name= "Weaver">PMID:9098893</ref>. These contrasting results raised the question: which of the two sites is the active site of the enzyme?
'''Fumarase C''' is an enzyme from Escherichia coli that catalyzes the stereospecific, reversible interconversion between L-malate and fumarate via the hydrolysis of the carbon-oxygen bond. The hydration/dehydration reaction catalyzed by fumarase C is of particular interest, because it plays an essential role in the metabolic citric acid cycle<ref>PMID:8909293</ref>. Fumarases from eukaryotic organisms have been studied and characterized to a much greater extent, compared to fumarase C from E. coli. In an attempt to further characterize EfumC, crystallographic studies were conducted using the enzyme, however the studies produced some unexpected results. Crystal structures of EfumC revealed that the enzyme has two dicarboxylate binding sites. More specifically, crystallographic studies that utilized inhibitors related to the enzyme’s normal substrate found that the inhibitors pyromellitic acid and β-trimethysilyl maleate bound to two different sites, which were named the A-site and B-site, respectfully<ref name= "Weaver">PMID:9098893</ref>. These contrasting results raised the question: which of the two sites is the active site of the enzyme?
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In order to answer this question, an experiment that tested each of the sites independently would need to be designed and conducted. Biochemical data suggested that a histidine side chain in both sites – <scene name='72/726367/His_188/1'>His 188</scene> in the A-site and <scene name='72/726367/His_129/1'>His 129</scene> in the B-site – was one of the key factors in substrate binding, so a mutation of the histidine residue in either of the sites would inhibit substrate binding. And, if the histidine residues were mutated independently, the mutation that dramatically affected the catalytic ability of the enzyme would show that the mutation was located in the active site of the enzyme. In order to experimentally test this hypothesis, two mutations were created: H188N and H129N. The two mutations were created by mutating a histidine at each of the sites into an asparagine. In order to quantify the effect of the mutations, the specific activity, average velocity, and average protein concentration of the reactions were measured and compared to wild-type EfumC. The results of the experiment showed that the H129N mutation had little effect on the enzymatic activity of the enzyme, as the specific activity of the enzyme was comparable to the wild-type enzyme. In contrast, the <scene name='72/726367/Ans_188_mutant/1'>H188N</scene> mutation dramatically reduced the specific activity of the catalytic reaction. These data strongly suggested that the H188 residue had a direct role in the catalytic mechanism of the enzyme and, therefore, that the H188 residue was located in the active site of the enzyme. Furthermore, it was concluded that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>.
In order to answer this question, an experiment that tested each of the sites independently would need to be designed and conducted. Biochemical data suggested that a histidine side chain in both sites – <scene name='72/726367/His_188/1'>His 188</scene> in the A-site and <scene name='72/726367/His_129/1'>His 129</scene> in the B-site – was one of the key factors in substrate binding, so a mutation of the histidine residue in either of the sites would inhibit substrate binding. And, if the histidine residues were mutated independently, the mutation that dramatically affected the catalytic ability of the enzyme would show that the mutation was located in the active site of the enzyme. In order to experimentally test this hypothesis, two mutations were created: H188N and H129N. The two mutations were created by mutating a histidine at each of the sites into an asparagine. In order to quantify the effect of the mutations, the specific activity, average velocity, and average protein concentration of the reactions were measured and compared to wild-type EfumC. The results of the experiment showed that the H129N mutation had little effect on the enzymatic activity of the enzyme, as the specific activity of the enzyme was comparable to the wild-type enzyme. In contrast, the <scene name='72/726367/Ans_188_mutant/1'>H188N</scene> mutation dramatically reduced the specific activity of the catalytic reaction. These data strongly suggested that the H188 residue had a direct role in the catalytic mechanism of the enzyme and, therefore, that the H188 residue was located in the active site of the enzyme. Furthermore, it was concluded that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>.

Current revision

Fumarase C from E. coli

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References

  1. ↑ Weaver T, Banaszak L. Crystallographic studies of the catalytic and a second site in fumarase C from Escherichia coli. Biochemistry. 1996 Nov 5;35(44):13955-65. PMID:8909293 doi:http://dx.doi.org/10.1021/bi9614702
  2. ↑ 2.0 2.1 2.2 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
  3. ↑ Weaver TM, Levitt DG, Donnelly MI, Stevens PP, Banaszak LJ. The multisubunit active site of fumarase C from Escherichia coli. Nat Struct Biol. 1995 Aug;2(8):654-62. PMID:7552727
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