Glycoside hydrolases - Revision history

Spencer Williams: /* NAD-dependent hydrolysis */

2025-06-24T06:37:57Z

NAD-dependent hydrolysis

Spencer Williams: /* Alternative nucleophiles */

2023-12-17T21:29:22Z

Alternative nucleophiles

Spencer Williams: /* References */

2023-12-17T21:28:59Z

References

Spencer Williams: /* Alternative nucleophiles */

2023-12-17T21:28:23Z

Alternative nucleophiles

Harry Brumer: /* By a neighboring 2-acetamido group */

2023-07-04T19:39:21Z

By a neighboring 2-acetamido group

Harry Brumer: tidied-up some links

2023-07-04T19:38:10Z

tidied-up some links

Spencer Williams at 23:57, 28 June 2023

2023-06-28T23:57:04Z

Spencer Williams: /* References */

2023-06-28T23:55:25Z

References

Spencer Williams: /* Neighboring group participation */

2023-06-28T23:54:52Z

Neighboring group participation

Spencer Williams: /* Neighboring group participation */

2023-06-28T23:54:13Z

Neighboring group participation

@@ Line 67: / Line 67: @@
 ====NAD-dependent hydrolysis====
-The members of [[GH4]], [[GH109]], [[GH177]] and [[GH179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-&beta;-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1<sub>cb</sub> elimination can occur with assistance from an enzymatic base. The &alpha;,&beta;-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.
+The members of [[GH4]], [[GH109]], [[GH177]], [[GH179]], and [[GH188]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-&beta;-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1<sub>cb</sub> elimination can occur with assistance from an enzymatic base. The &alpha;,&beta;-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.
 [[Image:Family_4_mechanism.png|center|700px]]

@@ Line 62: / Line 62: @@
 ====Alternative nucleophiles====
-Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn<sup>2+,/sup>-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.
+Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn<sup>2+</sup>-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.
 [[Image:sialidase_mechanism.png|center|700px]]

@@ Line 97: / Line 97: @@
 #Ndeh2017 pmid=28329766
 #Sobala2020 pmid=32490192
-#Thompson2012 pmid=22219371
+#McGregor2021 pmid=33528085
 </biblio>
 [[Category:Definitions and explanations]]

@@ Line 62: / Line 62: @@
 ====Alternative nucleophiles====
-Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.
+Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn<sup>2+,/sup>-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.
 [[Image:sialidase_mechanism.png|center|700px]]

@@ Line 49: / Line 49: @@
 ====Neighboring group participation====
 =====By a neighboring 2-acetamido group=====
-Enzymes of [[GH18]], [[GH20]], [[GH25]], [[GH56]], [[GH84]], [[GH85]] and [[GH123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzyme of glycoside hydrolase families [[Glycoside Hydrolase Family 3|3]] and [[Glycoside Hydrolase Family 22|22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.
+Enzymes of [[GH18]], [[GH20]], [[GH25]], [[GH56]], [[GH84]], [[GH85]] and [[GH123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzymes from [[GH3]] and [[GH22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.
 [[Image:Hex_neighboring_mechanism.png|centre|700px]]

@@ Line 54: / Line 54: @@
 '''2. By a neighboring 2-hydroxyl group'''<br>
-Enzymes of glycoside hydrolase family [[Glycoside Hydrolase Family 99|99]] hydrolyse a-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.
+Enzymes of glycoside hydrolase family [[Glycoside Hydrolase Family 99|99]] hydrolyse α-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.
 ====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
@@ Line 62: / Line 62: @@
 ====Alternative nucleophiles====
-Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-d-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.
+Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.
 [[Image:sialidase_mechanism.png|center|700px]]