Glycoside Hydrolase Family 34 - Revision history

Harry Brumer: Finally updated to Curator Approved after a long period of inactivity - page appears to be essentially complete

2023-01-05T01:00:37Z

Finally updated to Curator Approved after a long period of inactivity - page appears to be essentially complete

Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

2021-12-18T21:15:33Z

Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

Kyle Robinson at 17:44, 23 June 2020

2020-06-23T17:44:34Z

Kyle Robinson at 17:44, 23 June 2020

2020-06-23T17:44:11Z

Kyle Robinson at 17:38, 23 June 2020

2020-06-23T17:38:30Z

Kyle Robinson at 17:35, 23 June 2020

2020-06-23T17:35:06Z

Kyle Robinson at 17:28, 23 June 2020

2020-06-23T17:28:34Z

Kyle Robinson at 17:27, 23 June 2020

2020-06-23T17:27:01Z

Kyle Robinson at 17:25, 23 June 2020

2020-06-23T17:25:37Z

Kyle Robinson at 17:18, 23 June 2020

2020-06-23T17:18:20Z

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
-{{UnderConstruction}}
+{{CuratorApproved}}
 * [[Author]]: [[User:Kyle Robinson|Kyle Robinson]]
 * [[Responsible Curator]]:  [[User:Steve Withers|Steve Withers]]

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{UnderConstruction}}
-* [[Author]]: ^^^Kyle Robinson^^^
+* [[Author]]: [[User:Kyle Robinson|Kyle Robinson]]
-* [[Responsible Curator]]:  ^^^Steve Withers^^^
+* [[Responsible Curator]]:  [[User:Steve Withers|Steve Withers]]
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 == Catalytic Residues ==
 The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be directly involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9 of Neu5Ac. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>.  More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports the role of acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy [[Lexicon]] under [[General acid/base]].
 == Three-dimensional structures ==

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 == Three-dimensional structures ==
-These viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains. Homotetramers of these enzymes form mushroom-like structures on the surface of the virus <cite> Bovin2009 </cite>. Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes <cite> vonItzstein2007 </cite>. An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (<cite> KimWithers2013 Vavricka2013 </cite>.
+These viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains. Homotetramers of these enzymes form mushroom-like structures on the surface of the virus <cite> Bovin2009 </cite>. Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes <cite> vonItzstein2007 </cite>. An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available <cite> KimWithers2013 Vavricka2013 </cite>.
 == Family Firsts ==

← Older revision		Revision as of 17:38, 23 June 2020
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	== Catalytic Residues ==		== Catalytic Residues ==
−	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports the role of acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy [[Lexicon]] under [[General acid/base]].	+	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be directly involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9 of Neu5Ac. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports the role of acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy [[Lexicon]] under [[General acid/base]].

← Older revision		Revision as of 17:35, 23 June 2020
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	== Catalytic Residues ==		== Catalytic Residues ==
−	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to ~~Red Blood Cells~~ by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General ~~Acid~~/~~Base~~.	+	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports the role of acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy [[Lexicon]] under [[General acid/base]].

← Older revision		Revision as of 17:28, 23 June 2020
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	== Catalytic Residues ==		== Catalytic Residues ==
−	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) ~~render~~ interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.	+	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.

← Older revision		Revision as of 17:27, 23 June 2020
Line 37:		Line 37:

	== Catalytic Residues ==		== Catalytic Residues ==
−	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures showed that ~~this~~ glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.	+	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.

← Older revision		Revision as of 17:25, 23 June 2020
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	== Catalytic Residues ==		== Catalytic Residues ==
−	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276, though subsequent structures showed that this residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.	+	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures showed that this glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.

← Older revision		Revision as of 17:18, 23 June 2020
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	== Catalytic Residues ==		== Catalytic Residues ==
−	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same ~~paper~~ Asp151 was ~~considered~~ as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276, though subsequent structures showed that this residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.	+	The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276, though subsequent structures showed that this residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for Asp151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.