Fumarase 2 - Revision history

Ann Taylor at 19:56, 2 March 2023

2023-03-02T19:56:54Z

← Older revision		Revision as of 19:56, 2 March 2023
Line 10:		Line 10:

	== Active Site Characteristics ==		== Active Site Characteristics ==
	The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.		The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/6'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.
	</StructureSection>		</StructureSection>
	===References===		===References===
	<references/>		<references/>

Ann Taylor at 16:59, 28 January 2023

2023-01-28T16:59:49Z

← Older revision		Revision as of 16:59, 28 January 2023
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	<StructureSection load='1fuo' size='340' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)' scene = ''>		<StructureSection load='1fuo' size='340' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)' scene = ''>
	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>. Each subunit is comprised of <scene name='44/446278/Domains/1'>three domains</scene>.

	==Structure: will the real active site please stand?==		==Structure: will the real active site please stand?==

Ann Taylor at 02:56, 27 January 2023

2023-01-27T02:56:21Z

← Older revision		Revision as of 02:56, 27 January 2023
Line 1:		Line 1:
	<StructureSection load='1fuo' size='~~350~~' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>		==Fumarase==
			<StructureSection load='1fuo' size='340' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)' scene = ''>
	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.

	==Structure: will the real active site please stand?==		==Structure: will the real active site please stand?==
	Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?		Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?

	In order to answer this question, an experiment that tested each of the sites independently was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that 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 was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>.

Ann Taylor: Undo revision 3705943 by Ann Taylor (Talk)

2023-01-27T02:50:52Z

Undo revision 3705943 by Ann Taylor (Talk)

← Older revision		Revision as of 02:50, 27 January 2023
Line 1:		Line 1:
	<StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>		<StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>

	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.
Line 11:		Line 10:
	== Active Site Characteristics ==		== Active Site Characteristics ==
	The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.		The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.

	</StructureSection>		</StructureSection>

	===References===		===References===
	<references/>		<references/>

Ann Taylor at 02:48, 27 January 2023

2023-01-27T02:48:46Z

← Older revision		Revision as of 02:48, 27 January 2023
Line 1:		Line 1:
	<StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>		<StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>

	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.
Line 10:		Line 11:
	== Active Site Characteristics ==		== Active Site Characteristics ==
	The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.		The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.

	</StructureSection>		</StructureSection>

	===References===		===References===
	<references/>		<references/>

Ann Taylor at 02:47, 27 January 2023

2023-01-27T02:47:47Z

← Older revision		Revision as of 02:47, 27 January 2023
Line 1:		Line 1:
	<StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>		<StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'>
	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>.

	==Structure: will the real active site please stand?==		==Structure: will the real active site please stand?==
	~~Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits.~~ Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?		Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?

	In order to answer this question, an experiment that tested each of the sites independently was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that 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 was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>.

Ann Taylor: Undo revision 3705940 by Ann Taylor (Talk)

2023-01-27T02:25:13Z

Undo revision 3705940 by Ann Taylor (Talk)

← Older revision		Revision as of 02:25, 27 January 2023
Line 2:		Line 2:
	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.

	~~== Active Site Characteristics ==~~
	The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.

	==Structure: will the real active site please stand?==		==Structure: will the real active site please stand?==
	Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?		Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?
Line 11:		Line 7:
	In order to answer this question, an experiment that tested each of the sites independently was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that 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 was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>.

			== Active Site Characteristics ==
			The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.
	</StructureSection>		</StructureSection>
	===References===		===References===
	<references/>		<references/>

Ann Taylor at 02:24, 27 January 2023

2023-01-27T02:24:24Z

← Older revision		Revision as of 02:24, 27 January 2023
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	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.

			== Active Site Characteristics ==
			The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.

	==Structure: will the real active site please stand?==		==Structure: will the real active site please stand?==
	Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?		Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?
Line 7:		Line 11:
	In order to answer this question, an experiment that tested each of the sites independently was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that 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 was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. 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. This lead to the conclusion that that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>.

	~~== Active Site Characteristics ==~~
	The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.
	</StructureSection>		</StructureSection>
	===References===		===References===
	<references/>		<references/>

Karsten Theis at 13:45, 16 July 2020

2020-07-16T13:45:02Z

← Older revision		Revision as of 13:45, 16 July 2020
Line 8:		Line 8:

	== Active Site Characteristics ==		== Active Site Characteristics ==
	The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/2'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.		The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/3'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate.
	</StructureSection>		</StructureSection>
	===References===		===References===
	<references/>		<references/>

Karsten Theis at 14:33, 29 August 2019

2019-08-29T14:33:29Z

← Older revision		Revision as of 14:33, 29 August 2019
Line 2:		Line 2:
	===Overview===		===Overview===
	'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.		'''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa.
	==~~Stucture~~: will the real active site please stand?==		==Structure: will the real active site please stand?==
	Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?		Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site?