Sandbox Wabash 26 Fumarase - Revision history

Student at 20:39, 28 February 2016

Student — Sun, 28 Feb 2016 20:39:31 GMT

←Older revision		Revision as of 20:39, 28 February 2016
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	== Structural highlights ==		== Structural highlights ==

-	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen ~~binding~~ and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site. <scene name='72/726362/H129_mutant/1'>This structure</scene> shows the H129 mutant. <scene name='72/726362/Fumarase_quaternary_structure/2'>This structure</scene> shows the full quaternary structure of fumarase with all His129 and His188 labeled.	+	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen bonds to Ser98, Thr100 and Asn141 and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site. <scene name='72/726362/H129_mutant/1'>This structure</scene> shows the H129 mutant. <scene name='72/726362/Fumarase_quaternary_structure/2'>This structure</scene> shows the full quaternary structure of fumarase with all His129 and His188 labeled.

	</StructureSection>		</StructureSection>

Student at 20:37, 28 February 2016

Student — Sun, 28 Feb 2016 20:37:48 GMT

←Older revision		Revision as of 20:37, 28 February 2016
Line 3:		Line 3:
	This page is for Alex Waters' fumarase page.		This page is for Alex Waters' fumarase page.

-	<scene name='72/726362/Fumarase_nothing_bound/1'>Fumarase</scene> is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and <scene name='72/726362/His129/1'>His129</scene> would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.	+	<scene name='72/726362/Fumarase_nothing_bound/1'>Fumarase</scene> is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and <scene name='72/726362/His129/1'>His129</scene> would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the <scene name='72/726362/Active_site/1'>active site</scene>.
	== Function ==		== Function ==

Student at 19:48, 28 February 2016

Student — Sun, 28 Feb 2016 19:48:11 GMT

←Older revision		Revision as of 19:48, 28 February 2016
Line 12:		Line 12:
	== Structural highlights ==		== Structural highlights ==

-	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen binding and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site. <scene name='72/726362/Fumarase_quaternary_structure/2'>This structure</scene> shows the full quaternary structure of fumarase with all His129 and His188 labeled.	+	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen binding and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site. <scene name='72/726362/H129_mutant/1'>This structure</scene> shows the H129 mutant. <scene name='72/726362/Fumarase_quaternary_structure/2'>This structure</scene> shows the full quaternary structure of fumarase with all His129 and His188 labeled.

	</StructureSection>		</StructureSection>

Student at 19:40, 28 February 2016

Student — Sun, 28 Feb 2016 19:40:19 GMT

←Older revision		Revision as of 19:40, 28 February 2016
Line 12:		Line 12:
	== Structural highlights ==		== Structural highlights ==

-	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen binding and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site.	+	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen binding and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site. <scene name='72/726362/Fumarase_quaternary_structure/2'>This structure</scene> shows the full quaternary structure of fumarase with all His129 and His188 labeled.

	</StructureSection>		</StructureSection>

Student at 18:49, 28 February 2016

Student — Sun, 28 Feb 2016 18:49:25 GMT

←Older revision		Revision as of 18:49, 28 February 2016
Line 16:		Line 16:
	</StructureSection>		</StructureSection>
	== References ==		== References ==
-	~~<ref>9098893</ref>~~	+	Weaver, T., M. Lees, and L. Banaszak. "Mutations of Fumarase That Distinguish between the Active Site and a Nearby Dicarboxylic Acid Binding Site." PubMed. NCBI, 6 Apr. 1997. Web. 28 Feb. 2016.
	<references/>		<references/>

Student at 18:38, 28 February 2016

Student — Sun, 28 Feb 2016 18:38:41 GMT

←Older revision		Revision as of 18:38, 28 February 2016
Line 12:		Line 12:
	== Structural highlights ==		== Structural highlights ==

-	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen binding and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction.	+	The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen binding and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. <scene name='72/726362/H188_mutant_with_protein_bound/1'>This Structure</scene> shows the His188 mutant with L-malate bound to the putative active site.

	</StructureSection>		</StructureSection>

Student at 18:18, 28 February 2016

Student — Sun, 28 Feb 2016 18:18:04 GMT

←Older revision		Revision as of 18:18, 28 February 2016
Line 3:		Line 3:
	This page is for Alex Waters' fumarase page.		This page is for Alex Waters' fumarase page.

-	<scene name='72/726362/Fumarase_nothing_bound/1'>Fumarase</scene> is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and ~~H129~~ would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.	+	<scene name='72/726362/Fumarase_nothing_bound/1'>Fumarase</scene> is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and <scene name='72/726362/His129/1'>His129</scene> would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.
	== Function ==		== Function ==

Student at 17:45, 28 February 2016

Student — Sun, 28 Feb 2016 17:45:55 GMT

←Older revision		Revision as of 17:45, 28 February 2016
Line 3:		Line 3:
	This page is for Alex Waters' fumarase page.		This page is for Alex Waters' fumarase page.

-	Fumarase is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and H129 would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.	+	<scene name='72/726362/Fumarase_nothing_bound/1'>Fumarase</scene> is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and H129 would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.
	== Function ==		== Function ==

Student at 17:43, 28 February 2016

Student — Sun, 28 Feb 2016 17:43:11 GMT

←Older revision		Revision as of 17:43, 28 February 2016
Line 3:		Line 3:
	This page is for Alex Waters' fumarase page.		This page is for Alex Waters' fumarase page.

-	Fumarase is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. H188 was expected to change catalytic activity if the A-site was the active site and H129 would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.	+	Fumarase is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. <scene name='72/726362/His188/1'>H188</scene> was expected to change catalytic activity if the A-site was the active site and H129 would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the active site.
	== Function ==		== Function ==

Student at 17:35, 28 February 2016

Student — Sun, 28 Feb 2016 17:35:02 GMT

←Older revision		Revision as of 17:35, 28 February 2016
Line 16:		Line 16:
	</StructureSection>		</StructureSection>
	== References ==		== References ==
		+	<ref>9098893</ref>
	<references/>		<references/>