Sandbox Wabash 02 Fumarase
From Proteopedia
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==Determination of the True Active Site of Fumarase C from E. Coli== | ==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. | + | 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> |
==Structure of the Active Site of Fumarase== | ==Structure of the Active Site of Fumarase== | ||
| - | A | + | 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> | ||
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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. <ref>PMID:9098893</ref> | ||
| - | 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. <ref>PMID:9098893</ref> | ||
| - | Removal of the carboxylate binding site is due to a rotation of N129 about x, compared to the imidazole side chain position in the native structure. N129 is stabilized in this new position by forming bifurcated hydrogen bonds between its OD1 atom and the backbone nitrogen atoms of N131 and D132. These two backbone atoms form important hydrogen bonds to the O1 and O2 atoms of the dicarboxylic acid at the B-site in the native enzyme but are blocked by internal H-bonds in the H 129N mutant. The carboxyamide side chain of N129 in the mutant behaves like the carboxylate of ligand bound at site B. <ref>PMID:9098893</ref> | ||
| - | To further confirm that dicarboxylic acids will no longer bind to the B-site due to the steric hindrance imposed by N129, an H129N crystal was soaked in a solution containing P-trimethylsilyl maleate (TMSM) under the exact conditions used to generate the complex reported by Weaver and Banaszak (1996). No binding of TMSM was visible in the resulting maps. This result not only proved unambiguously that the A-site was the active site, but also that the H129N mutation dramatically reduced binding at the B-site. The A-site or active site in the crystal structure of H129N was unchanged by the mutation at H129. <ref>PMID:9098893</ref> | ||
| - | The mutation at the A-site, H188N, had a dramatic effect on the fumarase activity and suggested that this histidine side chain may have a direct role in the catalytic mechanism in H188N. The absence of the histidine side chain effectively reduces binding of citrate so that it is missing in the electron density maps. Another observation with respect to the H188N mutation is that the active site water molecule (W-26) is still present although shifted by approximately 0.70 A compared to the crystal structure of the wild-type enzyme. In the crystal structure of H188N, W-26 makes four hydrogen bonds to protein atoms. The replacement asparagine side chain, N188, forms two hydrogen bonds to W-26, one of which is rather short- 2.59 A. The N188 side chain seems able to mimic that of H188, leading to the previously observed short hydrogen bond between the side chain at 188 and W-26. S98b and N141b form the other two hydrogen bonds to the active site water. T100-OGI, which is one of the reported atoms interacting with the water in the crystal structure of native fumarase is no longer within hydrogen bonding distance. In the wild type, the active site water W-26 is hydrogen bonded to five different atoms. There are four protein ligands from two subunits including residues T1OOb, S98b, N141b. and H188c. The other interaction is only formed when a competitive inhibitor is present. With bound citrate ion, the water interaction involves the carboxylate at the C1 position (Weaver & Banaszak, 1996). In the case of the H129N structure, W-26 maintains all five of the interactions. <ref>PMID:9098893</ref> | ||
== <ref>PMID:9098893</ref>== | == <ref>PMID:9098893</ref>== | ||
Revision as of 00:46, 26 April 2016
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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]
