Sandbox Wabash 02 Fumarase
From Proteopedia
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| - | + | <Structure load='1YFE' size='300' color='white' frame='true' align='right' caption='testcaption' /> | |
| - | < | + | ==Determination of the True Active Site of Fumarase C from E. Coli== |
| - | + | Fumarase C from E. coli is an enzyme homologous to the cytosolic and mitochondrial enzymes found in eukaryotic cells. It catalyzes the hydration/dehydration reaction between the metabolites L-malate and fumarate. Fumarase C is a tetrameric enzyme composed of four identical sub-units of 50 kDa each while each subunit is mostly composed of α- helices. Previous studies have found that fumarase has two different carboxylic acid binding sites (<scene name='72/726360/Wild-type_a-site/1'>Wild-Type A-Site</scene> and <scene name='72/726360/Wild-type_b-site/2'>Wild-Type B-Site</scene>) in both wild-type and mutated forms of the enzyme. Results from these studies led to the dilemma as to which of the two sites was the active site. Because the A site is deeper at the center of fumarase and involves residues from three of the four subunits, it received initial support to be the true active site. Further experiments were done to determine which of these binding sites was the true active site. The H129N <scene name='72/726360/2fus/1'>H129N with bound Citrate</scene> and H188N <scene name='72/726360/H188n_with_bound_l-malate/1'>H188N with bound L-malate at activation site</scene> mutants were generated to resolve the two site problem. If the A-site was the active site, changing H188 should dramatically affect the catalytic activity. Conversely, if the B-site was the active site then a mutation at H129 should affect catalysis. A histidine to asparagine mutation at the A site resulted in a large decrease in specific activity, whereas a histidine to asparagine mutation at the B site resulted in essentially no effect. Therefore, it was determined that the A site is the active site, although the B site binds to substrates and their analogs as well with a lower ligand binding affinity.<ref>PMID:9098893</ref> | |
| - | Fumarase C from E. coli is an enzyme homologous | + | |
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| - | + | ==Structure of the Active Site of Fumarase== | |
| + | Eukaryotic fumarase catalysis kinetics, stereochemical characteristics and mutant kinetics have been extensively studied. The determined active A site contains residues of Asn 141, Thr 100, Glu 331, Ser 98, His 188, Asn 326 and Lys 324. Asn 141 is especially important because it is one of the amino acids that forms hydrogen bonds with water in the active site. Water and this residue are vital to the enzyme’s mechanism and function. | ||
| + | Site A is in a relatively deep pit removed from bulk solvent and contained a bound water. One of the side chains interacting with this water molecule is H188. In the crystallographic coordinates of the wild-type enzyme, the water molecule forms a short hydrogen bond, 2.5 A, with the imidazole ring of H188. The side chain of H188 is also within hydrogen bonding distance to an oxygen atom of bound citrate or pyromellitic acid. The B-site is closer to the surface of the enzyme. There are three principal interactions between the ligand and wild-type fumarase at the B-site, and the A- and B-sites are linked by residues 131 to 140 in a single subunit. Main chain hydrogen bonds between the oxygen atoms of the bound ligand and main chain -NHs of D132 and N131 on the N-terminal end of the pi-helix are important to stabilization at the B-site. Oxygen atoms of the other carboxylate of the ligand at the B-site are hydrogen bonded to R126-NE and H129-NDI. The hydrogen bonds between side chain atoms of N135 and N103, and between N103 and S140 form an indirect connection between the B- and the A-site. H129 is the only basic group close to a ligand bound at the B-site.<ref>PMID:9098893</ref> | ||
| - | As evident from the calculated specific activities for wild-type fumarase and the H129N and the H188N mutants, little effect is observed in changing H129 into an asparagine residue as both wild type and H129N have about the same specific activity. However, the H188N mutation dramatically affects the catalytic reaction. The specific activity of the H188N is approximately 200 times less than that of wild-type enzyme or H129N. These results prove that the active site of fumarase is near H188 or the A-site. The significance of the B-site to the catalytic reaction is unknown. X-ray crystal structures were determined to see what effects the mutations had on local conformation at both the A- and B-sites. | ||
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| - | '''Structure of the Active Site of Fumarase'''. | ||
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| - | A number of stereochemical factors describing the two sites were examined in the wild-type crystal structures (Weaver & Banaszak, 1996). Site A was in a relatively deep pit removed from bulk solvent. It also contained an unusual bound water, although there was no obvious way of linking this directly to the catalytic process. One of the side chains interacting with this water molecule is H188. In the crystallographic coordinates of the wildtype enzyme, the water molecule forms a short hydrogen bond, 2.5 A, with the imidazole ring of H188. The side chain of H188 is also within hydrogen bonding distance to an oxygen atom of bound citrate or pyromellitic acid. The B-site is closer to the surface of the enzyme (Weaver & Banaszak, 1996). There are three principal interactions between the ligand and wild-type fumarase at the B-site, and in an indirect fashion the A- and B-sites are linked by residues 131 to 140 in a single subunit. Main chain hydrogen bonds between the oxygen atoms of the bound ligand and main chain -NHs of D132 and N131 on the N-terminal end of the pi-helix are important to stabilization at the B-site. Oxygen atoms of the other carboxylate of the ligand at the B-site are hydrogen bonded to R126-NE and H129-NDI. The hydrogen bonds between side chain atoms of N135 and N103, and between N103 and S140 form an indirect connection between the B- and the A-site. H129 is the only basic group close to a ligand bound at the B-site. | ||
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| - | Values for phi and psi angles were plotted and compared with normally allowed torsional regions (Laskowski et al., 1993). Out of a total of 911 residues, only F356 fell outside of the allowed region. It is surrounded by a number of additional hydrophobic residues from three of the four subunits within the tetramer. F356b belongs to a sharp turn with the side chain pointing into a hydrophobic pocket that lies behind the active site. It is near to one of the molecular dyads and therefore close to both W297c and W297d. It is also positioned near L358b, which is close to the active site water molecule and it has van der Waal contacts with R186c and H 188c, both of which are considered part of the active site. Other residues within van der Waal contact of F356b are from the c-subunit including L298c, I306c, and L189c. Although attention was drawn to this phenylalanine by its unusual phi and psi angles, it is clearly a pivotal residue at the juncture of subunits near the active site. | ||
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| - | The H129N mutation was made to characterize the functional significance of a dicarboxylic acid binding site we have labeled the B-site. The B-site is formed from a single subunit of the tetramer and includes atoms from residues R126, H129, N131, and D132 (Weaver & Banaszak, 1996). H129 is the only potential side | ||
| - | chain that could serve as one of the catalytic bases in the B-site (Brant et al., 1963). The H129N mutation had little effect on catalytic activity, confirming the active site to be site A. The crystal structure of H129N showed that the mutated protein had essentially the same conformation as the wild type but appeared to dramatically reduce binding of ligands at the B-site. | ||
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| - | You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue. | ||
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| - | == Function == | ||
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| - | == Disease == | ||
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| - | == Relevance == | ||
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| - | == Structural highlights == | ||
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| - | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. | ||
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| - | </StructureSection> | ||
== References == | == References == | ||
<references/> | <references/> | ||
Current revision
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Determination of the True Active Site of Fumarase C from E. Coli
Fumarase C from E. coli is an enzyme homologous to the cytosolic and mitochondrial enzymes found in eukaryotic cells. It catalyzes the hydration/dehydration reaction between the metabolites L-malate and fumarate. Fumarase C is a tetrameric enzyme composed of four identical sub-units of 50 kDa each while each subunit is mostly composed of α- helices. Previous studies have found that fumarase has two different carboxylic acid binding sites ( and ) in both wild-type and mutated forms of the enzyme. Results from these studies led to the dilemma as to which of the two sites was the active site. Because the A site is deeper at the center of fumarase and involves residues from three of the four subunits, it received initial support to be the true active site. Further experiments were done to determine which of these binding sites was the true active site. The H129N and H188N mutants were generated to resolve the two site problem. If the A-site was the active site, changing H188 should dramatically affect the catalytic activity. Conversely, if the B-site was the active site then a mutation at H129 should affect catalysis. A histidine to asparagine mutation at the A site resulted in a large decrease in specific activity, whereas a histidine to asparagine mutation at the B site resulted in essentially no effect. Therefore, it was determined that the A site is the active site, although the B site binds to substrates and their analogs as well with a lower ligand binding affinity.[1]
Structure of the Active Site of Fumarase
Eukaryotic fumarase catalysis kinetics, stereochemical characteristics and mutant kinetics have been extensively studied. The determined active A site contains residues of Asn 141, Thr 100, Glu 331, Ser 98, His 188, Asn 326 and Lys 324. Asn 141 is especially important because it is one of the amino acids that forms hydrogen bonds with water in the active site. Water and this residue are vital to the enzyme’s mechanism and function. Site A is in a relatively deep pit removed from bulk solvent and contained a bound water. One of the side chains interacting with this water molecule is H188. In the crystallographic coordinates of the wild-type enzyme, the water molecule forms a short hydrogen bond, 2.5 A, with the imidazole ring of H188. The side chain of H188 is also within hydrogen bonding distance to an oxygen atom of bound citrate or pyromellitic acid. The B-site is closer to the surface of the enzyme. There are three principal interactions between the ligand and wild-type fumarase at the B-site, and the A- and B-sites are linked by residues 131 to 140 in a single subunit. Main chain hydrogen bonds between the oxygen atoms of the bound ligand and main chain -NHs of D132 and N131 on the N-terminal end of the pi-helix are important to stabilization at the B-site. Oxygen atoms of the other carboxylate of the ligand at the B-site are hydrogen bonded to R126-NE and H129-NDI. The hydrogen bonds between side chain atoms of N135 and N103, and between N103 and S140 form an indirect connection between the B- and the A-site. H129 is the only basic group close to a ligand bound at the B-site.[2]
References
- ↑ 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
