Glycoside Hydrolase Family 20 - Revision history

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

2021-12-18T21:20:17Z

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

Harry Brumer: /* References */ Minor DOI link reformatting

2013-09-23T23:06:47Z

References: Minor DOI link reformatting

Spencer Williams: /* Family Firsts */

2011-03-10T09:11:14Z

Family Firsts

Spencer Williams: /* Substrate specificities */

2011-03-10T09:09:56Z

Substrate specificities

Spencer Williams: /* Substrate specificities */

2011-03-10T09:09:30Z

Substrate specificities

Spencer Williams: /* Family Firsts */

2011-03-10T08:52:05Z

Family Firsts

Spencer Williams: /* Substrate specificities */

2011-03-10T08:50:47Z

Substrate specificities

Spencer Williams at 08:50, 10 March 2011

2011-03-10T08:50:03Z

Harry Brumer: /* References */ Japanese refs. from pmids

2011-02-25T08:30:09Z

References: Japanese refs. from pmids

Harry Brumer: /* References */ Added DOI links

2011-02-25T08:26:49Z

References: Added DOI links

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 <!-- CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]: ^^^Ian Greig^^^
+* [[Author]]: [[User:Ian Greig|Ian Greig]]
-* [[Responsible Curator]]:  ^^^David Vocadlo^^^
+* [[Responsible Curator]]:  [[User:David Vocadlo|David Vocadlo]]
 ----

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 #Yamamoto74 pmid=4426886
 #Mega70 pmid=5452757
-#Lowe67 Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) Lysozyme-Catalysed Hydrolysis of some 'β''-Aryl Di-''N''-acetylchitobiosides. Biochem J. 104(3), 893-899.  //''Link to article:'' [http://www.biochemj.org/bj/104/bj1040893.htm ''Biochem. J.'']
+#Lowe67 Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) ''Lysozyme-Catalysed Hydrolysis of some ''β''-Aryl Di-''N''-acetylchitobiosides.'' Biochem J. 104(3), 893-899.  [http://www.biochemj.org/bj/104/bj1040893.htm Article online.]
-#Sinnott76 Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620. //''Link to article:'' [http://dx.doi.org/10.1039/P29760000618 DOI:10.1039/P29760000618]
+#Sinnott76 Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620. [http://dx.doi.org/10.1039/P29760000618 DOI:10.1039/P29760000618]
-#Bruice67 Piszkiewicz, D, Bruice, T (1967) Glycoside Hydrolysis. I. Intramolecular Acetamido and Hydroxyl Group Catalysis in Glycoside Hydrolysis. J. Am. Chem. Soc. 89, 6237-6243.  //''Link to article:'' [http://dx.doi.org/10.1021/ja01000a044 DOI:10.1021/ja01000a044]
+#Bruice67 Piszkiewicz, D, Bruice, T (1967) Glycoside Hydrolysis. I. Intramolecular Acetamido and Hydroxyl Group Catalysis in Glycoside Hydrolysis. J. Am. Chem. Soc. 89, 6237-6243.  [http://dx.doi.org/10.1021/ja01000a044 DOI:10.1021/ja01000a044]
-#Bruice68_1 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. II. Intramolecular Carboxyl and Acetamido Group Catalysis in β-Glycoside Hydrolysis. J. Am. Chem. Soc. 90, 2156-2163. //''Link to article:'' [http://dx.doi.org/10.1021/ja01010a038 DOI:10.1021/ja01010a038]
+#Bruice68_1 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. II. Intramolecular Carboxyl and Acetamido Group Catalysis in β-Glycoside Hydrolysis. J. Am. Chem. Soc. 90, 2156-2163. [http://dx.doi.org/10.1021/ja01010a038 DOI:10.1021/ja01010a038]
-#Bruice68_2 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. III. Intramolecular Acetamido Group Participation in the Specific Acid Catalyzed Hydrolysis of Methyl-2-Acetamido-2-deoxy-''β''-D-glucopyranoside. J. Am. Chem. Soc. 90, 5844-5848. //''Link to article:'' [http://dx.doi.org/10.1021/ja01023a032 DOI:10.1021/ja01023a032]
+#Bruice68_2 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. III. Intramolecular Acetamido Group Participation in the Specific Acid Catalyzed Hydrolysis of Methyl-2-Acetamido-2-deoxy-''β''-D-glucopyranoside. J. Am. Chem. Soc. 90, 5844-5848. [http://dx.doi.org/10.1021/ja01023a032 DOI:10.1021/ja01023a032]
 #Kosman80 pmid=7440573
-#Knapp96 Knapp, S, Vocadlo, DJ, Gao, Z, Kirk, B, Lou, J, Withers, SG (1996) NAG-thiazoline, An ''N''-Acetyl-''β''-hexosaminidase Inhibitor That Implicates Acetamido Participation. J. Am. Chem. Soc. 118, 6804-6805. //''Link to article:'' [http://dx.doi.org/10.1021/ja960826u DOI:10.1021/ja960826u]
+#Knapp96 Knapp, S, Vocadlo, DJ, Gao, Z, Kirk, B, Lou, J, Withers, SG (1996) NAG-thiazoline, An ''N''-Acetyl-''β''-hexosaminidase Inhibitor That Implicates Acetamido Participation. J. Am. Chem. Soc. 118, 6804-6805. [http://dx.doi.org/10.1021/ja960826u DOI:10.1021/ja960826u]
 #Mahuran04 pmid=14724290
 #DJV2005 pmid=15795231

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 == Family Firsts ==
 ;First sterochemistry determination:   The first determination of [[retaining]] stereochemistry for a known member of GH20 was on the ''Serratia marscescens'' enzyme <cite>Armand1997</cite>. The stereochemistry of hydrolysis of three different hexosaminidases (human placenta, jack bean, and bovine kidney) was shown by the Withers group in 1994  <cite>Lai</cite> and it is now generally assumed that some of these are GH20 enzymes.
-;First [[catalytic nucleophile]] identification: These enzymes employ neighbouring group participation using the substrate ''N''-acetyl group. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae'' <cite>Mega70</cite> and ''Aspergillus niger'' <cite>Kosman80</cite> supported such a mechanism. This mechanism was further suggested by both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with [[GH18]] enzymes), through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>, and by potent inhibition of Jack Bean ''β''-hexosaminidase by NAG-thiazoline <cite>Knapp96</cite>.
+;First [[catalytic nucleophile]] identification: These enzymes employ neighbouring group participation using the substrate ''N''-acetyl group. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae'' <cite>Mega70</cite> and ''Aspergillus niger'' <cite>Kosman80</cite> supported such a mechanism. This mechanism was suggested by analysis of both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with [[GH18]] enzymes), through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>, and by potent inhibition of jack bean ''β''-hexosaminidase by NAG-thiazoline <cite>Knapp96</cite>.
 ;First [[general acid/base]] residue identification: Inferred from the 3-D structure <cite>Tews1996</cite> and by analogy with structurally related GH18 chitinases.
 ;First 3-D structure: The 3-D structure of the ''Serratia marscescens'' chitobiase <cite>Tews1996</cite>.

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 == Substrate specificities ==
-GH20 [[glycoside hydrolases]] comprise both eukaryotic and prokaryotic enzymes. In addition to ''exo''-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO<sub>3</sub>-''N''-acetylglucosaminidases, GH20 also contains ''[[exo]]''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of GH20 are the human isoenzymes hexosaminidase A (a heterodimer of ''&alpha;'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the G<sub>M2</sub> ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders <cite>Mahuran04</cite>. [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]
+GH20 [[glycoside hydrolases]] comprise both eukaryotic and prokaryotic enzymes. In addition to ''[[exo]]''-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO<sub>3</sub>-''N''-acetylglucosaminidases, GH20 also contains ''[[exo]]''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of GH20 are the human isoenzymes hexosaminidase A (a heterodimer of ''&alpha;'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the G<sub>M2</sub> ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders <cite>Mahuran04</cite>. [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]
 == Kinetics and Mechanism ==

@@ Line 29: / Line 29: @@
 == Substrate specificities ==
-GH20 [[glycoside hydrolases]] comprise both eukaryotic and prokaryotic enzymes. In addition to ''exo''-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO<sub>3</sub>-''N''-acetylglucosaminidases, GH20 also contains [[''exo'']]-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of GH20 are the human isoenzymes hexosaminidase A (a heterodimer of ''&alpha;'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the G<sub>M2</sub> ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders <cite>Mahuran04</cite>. [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]
+GH20 [[glycoside hydrolases]] comprise both eukaryotic and prokaryotic enzymes. In addition to ''exo''-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO<sub>3</sub>-''N''-acetylglucosaminidases, GH20 also contains ''[[exo]]''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of GH20 are the human isoenzymes hexosaminidase A (a heterodimer of ''&alpha;'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the G<sub>M2</sub> ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders <cite>Mahuran04</cite>. [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]
 == Kinetics and Mechanism ==

@@ Line 29: / Line 29: @@
 == Substrate specificities ==
-GH20 members comprise enzymes from both eukaryotes and prokaryotes. In addition to exo-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO<sub>3</sub>-''N''-acetylglucosaminidases, GH20 also contains ''exo''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of this family are the human isoenzymes hexosaminidase A (a heterodimer of ''&alpha;'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the G<sub>M2</sub> ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders <cite>Mahuran04</cite>. [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]
+GH20 [[glycoside hydrolases]] comprise enzymes from both eukaryotes and prokaryotes. In addition to ''exo''-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO<sub>3</sub>-''N''-acetylglucosaminidases, GH20 also contains [[''exo'']]-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of GH20 are the human isoenzymes hexosaminidase A (a heterodimer of ''&alpha;'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the G<sub>M2</sub> ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders <cite>Mahuran04</cite>. [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]
 == Kinetics and Mechanism ==
-Neighbouring group participation has long been established as a reasonable mechanism for glycoside hydrolysis in solution<cite>Sinnott76 Bruice67 Bruice68_1 Bruice68_2</cite> and originally outlined as a possible, though subsequently refuted, mechanism for the hen egg-white lysozyme-catalyzed cleavage of ''β''-aryl di-''N''-acetylchitobiosides <cite>Lowe67</cite>. The earliest kinetic evidence supporting a mechanism involving neighbouring group participation in an enzyme-catalyzed hydrolysis <cite>Yamamoto73 Yamamoto74</cite> can be found for an ''N''-acetyl-''β''-D-glucosaminidase isolated from ''Aspergillus oryzae'' <cite>Mega70</cite>, likely a GH20 enzyme. This work used free energy relationships to infer neighbouring group participation although complete Michaelis-Menten kinetic parameters were not determined. Such kinetic parameters were determined for a ''β''-''N''-acetylglucosaminidase from ''Aspergillus niger'' and a similar free energy relationship-based analysis carried out to infer neighbouring group participation for this enzyme which, though unknown, is likely from GH20 <cite>Kosman80</cite>. The potency of "NAG-thiazoline" as a competitive inhibitor of the jack bean ''N''-acetyl-''β''-D-hexosaminidase (''K''<sub>i</sub> = 280 nM) has also been used to infer a mechanism of neighbouring group participation although, interestingly, the only retaining hexosaminidases reported currently (November 2010) reported in the CAZy database for the genus ''Canavalia'' are found in [[GH18]] <cite>Knapp96</cite>.
+GH20 enzymes are [[retaining]] [[glycoside hydrolases]] <cite>Armand1997</cite>. [[Neighboring group participation]] has long been established as a reasonable mechanism for glycoside hydrolysis in solution<cite>Sinnott76 Bruice67 Bruice68_1 Bruice68_2</cite> and originally outlined as a possible, though subsequently refuted, mechanism for the hen egg-white lysozyme-catalyzed cleavage of ''β''-aryl di-''N''-acetylchitobiosides <cite>Lowe67</cite>. The earliest kinetic evidence supporting a mechanism involving [[neighboring group participation]] in an enzyme-catalyzed hydrolysis <cite>Yamamoto73 Yamamoto74</cite> can be found for an ''N''-acetyl-''β''-D-glucosaminidase isolated from ''Aspergillus oryzae'' <cite>Mega70</cite>, likely a GH20 enzyme. This work used free energy relationships to infer neighbouring group participation although complete Michaelis-Menten kinetic parameters were not determined. Such kinetic parameters were determined for a ''β''-''N''-acetylglucosaminidase from ''Aspergillus niger'' and a similar free energy relationship-based analysis carried out to infer neighbouring group participation for this enzyme which, though unknown, is likely from GH20 <cite>Kosman80</cite>. The potency of "NAG-thiazoline" as a competitive inhibitor of the jack bean (''Canavalia'' spp) ''N''-acetyl-''β''-D-hexosaminidase (''K''<sub>i</sub> = 280 nM) has also been used to infer neighboring group participation in the catalytic mechanism although, interestingly, the only [[retaining]] hexosaminidases reported currently (November 2010) in the CAZy database for the genus ''Canavalia'' are found in [[GH18]] <cite>Knapp96</cite>.
 Deacetylation of the non-reducing end of a series of chito-oligosaccharides results in a loss of activity of ''Serratia marscescens'' chitobiase, an established GH20 enzyme, towards these compounds, which instead act as competitive inhibitors <cite>Armand1997</cite>. Moreover the structure of ''Serratia marcescens'' chitobiase in complex with a substrate provides structural support for a substrate-assisted mechanism. [[Image:GH20inhibitors.jpg|thumb|450px|'''Competitive Inhibitors of hexosaminidases.''' "NAG-thiazoline" (upper panel) and non-reducing end deacetylated chito-oligosaccharides are competitive inhibitors of hexosamindase employing neighbouring group participation]]  A comparative analysis of the activity of ''Streptomyces plicatus'' ''β''-hexosaminidase (SpHex, GH20) and ''Vibrio furnisii'' ''β''-hexosaminidase (ExoII, GH3) towards ''p''-nitrophenyl ''N''-acyl glucosaminides highlights contrasting reactivity trends expected for families of ''β''-glucosaminidase utilizing a mechanism of substrate-assisted catalysis (GH20) and those which do not ([[GH3]]): sharp decreases in activity with increasing ''N''-acyl fluorination are observed in the case of the SpHex enzyme whereas negligible changes in activity are observed for ExoII <cite>SGW05</cite>.
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 == Catalytic Residues ==
-The key catalytic residues of GH20 enzymes are found in conserved D-E amino acid pair. This catalytic diad is preceded in the primary sequence by the consensus H-x-G-G motif. The glutamate residue functions as the catalytic general acid/base. As these enzymes employ neighbouring group participation the preceding aspartate is not a nucleophile. Rather kinetic and crystallographic studies have shown that this residue orients and polarizes the catalytic ''N''-acetyl residue <cite>SJW2002</cite>. It may function either as a general base by deprotonating the ''N''-acetyl group in the intermediate and forming a neutral oxazoline intermediate, or alternatively it may electrostatically stabilize a positively charge oxazolinium ion intermediate.  The catalytic ''N''-acetyl group of the substrate is bound in a hydrophobic pocket defined by three conserved tryptophan residues. These three tryptophan residues define a compact pocket which does not accommodate (non-native) extended ''N''-acyl side-chains as readily as the elongated hydrophobic pocket found in GH84 enzymes <cite>DJV2005</cite>.
+The key catalytic residues of GH20 enzymes are found in a conserved D-E amino acid pair. This catalytic diad is preceded in the primary sequence by the consensus H-x-G-G motif. The glutamate residue functions as the catalytic general acid/base. As these enzymes employ neighbouring group participation the preceding aspartate is not a nucleophile. Rather kinetic and crystallographic studies have shown that this residue orients and polarizes the ''N''-acetyl residue <cite>SJW2002</cite>. It may function either as a [[general base]] by deprotonating the ''N''-acetyl group in the intermediate and forming a neutral oxazoline [[intermediate]], or alternatively it may electrostatically stabilize a positively charge [[oxazolinium ion]] [[intermediate]].  The ''N''-acetyl group of the substrate is bound in a hydrophobic pocket defined by three conserved tryptophan residues. These three tryptophan residues define a compact pocket which does not accommodate (non-native) extended ''N''-acyl side-chains as readily as the elongated hydrophobic pocket found in [[GH84]] enzymes <cite>DJV2005</cite>.
 == Three-dimensional structures ==
@@ Line 46: / Line 46: @@
 == Family Firsts ==
 ;First sterochemistry determination:   The first stereochemical determination for a known member of GH20 was on the ''Serratia marscescens'' enzyme <cite>Armand1997</cite>. The stereochemistry of hydrolysis of three different hexosaminidases (human placenta, jack bean, and bovine kidney) was shown by the Withers group in 1994  <cite>Lai</cite> and it is now generally assumed that some of these are GH20 enzymes.
-;First catalytic nucleophile identification: These enzymes employ neighbouring group participation. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae'' <cite>Mega70</cite> and ''Aspergillus niger'' <cite>Kosman80</cite> supported such a mechanism. This mechanism was further suggested by both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with [[GH18]] enzymes), through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>, and by potent inhibition of Jack Bean ''β''-hexosaminidase by NAG-thiazoline <cite>Knapp96</cite>.
+;First [[catalytic nucleophile]] identification: These enzymes employ neighbouring group participation using the substrate ''N''-acetyl group. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae'' <cite>Mega70</cite> and ''Aspergillus niger'' <cite>Kosman80</cite> supported such a mechanism. This mechanism was further suggested by both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with [[GH18]] enzymes), through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>, and by potent inhibition of Jack Bean ''β''-hexosaminidase by NAG-thiazoline <cite>Knapp96</cite>.
-;First general acid/base residue identification: Inferred from the 3-D structure <cite>Tews1996</cite> and by analogy with structurally related GH18 chitinases.
+;First [[general acid/base]] residue identification: Inferred from the 3-D structure <cite>Tews1996</cite> and by analogy with structurally related GH18 chitinases.
 ;First 3-D structure: The 3-D structure of the ''Serratia marscescens'' chitobiase <cite>Tews1996</cite>.

@@ Line 52: / Line 52: @@
 == References ==
 <biblio>
-#Yamamoto73 Yamamoto, K, (1973) ''N''-Acyl Specificity of Taka-N-acetyl-''β''-D-glucosaminidase Studied by Synthetic Substrate Analogs II. Preparation of Some ''p''-Nitrophenyl 2-Halogenoacetylamino-2-deoxy-''β''-D-glucopyranoside and Their Susceptibility to Enzymic Hydrolysis. J. Biochem. 73, 749-753.
+#Yamamoto73 pmid=4720059
-#Yamamoto74 Yamamoto, K, (1974) A Quantitative Approach to the Evaluation of ''β''-Acetamide Substituent Effects on the Hydrolysis by Taka-N-acetyl-''β''-D-glucosaminidase. Role of the Substrate 2-Acetamide Group in the ''N''-Acyl Specificity of the Enzyme J. Biochem. 76, 385-390. //''Link to article:'' [http://www.biochemj.org/bj/104/bj1040893.htm ''Biochem. J.'']
+#Yamamoto74 pmid=4426886
-#Mega70 Mega, T, Ikenaka, T, Matsushima, Y, (1970) Studies on ''N''-Acetyl-''β''-D-glucosaminidase of ''Aspergillus oryzae''. J. Biochem. 68, 109-117.
+#Mega70 pmid=5452757
 #Lowe67 Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) Lysozyme-Catalysed Hydrolysis of some 'β''-Aryl Di-''N''-acetylchitobiosides. Biochem J. 104(3), 893-899.  //''Link to article:'' [http://www.biochemj.org/bj/104/bj1040893.htm ''Biochem. J.'']
 #Sinnott76 Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620. //''Link to article:'' [http://dx.doi.org/10.1039/P29760000618 DOI:10.1039/P29760000618]