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Difference between revisions of "Glycoside Hydrolase Family 26"
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Family GH26 enzymes are “retainers”, as shown by NMR and follow a classical Koshland double-displacement mechanism. Pre-steady state kinetics using activated substrates revealed the two phases of the reaction; the rapid initial glycosylation step (only with good leaving groups) followed by the slower deglycosylation. It should be noted that the use of substrates with a good leaving group result in a very low apparent KM, particularly with the acid-base mutant. This does not reflect tight affinity but simply that the glycosylation step (k2) is much quicker than the deglycosylation step (k3) {Bolam, 1996 #7}. | Family GH26 enzymes are “retainers”, as shown by NMR and follow a classical Koshland double-displacement mechanism. Pre-steady state kinetics using activated substrates revealed the two phases of the reaction; the rapid initial glycosylation step (only with good leaving groups) followed by the slower deglycosylation. It should be noted that the use of substrates with a good leaving group result in a very low apparent KM, particularly with the acid-base mutant. This does not reflect tight affinity but simply that the glycosylation step (k2) is much quicker than the deglycosylation step (k3) {Bolam, 1996 #7}. | ||
| − | == Catalytic Residues == | + | == Catalytic Residues ==\ Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 |
| + | |||
| + | The catalytic residues were first identified in the endo-beta1,4-mannanase CjMan26A. The catalytic acid-base is the glutamate Glu320, which is separated in sequence by ~100 residues from the catalytic nucleophile, Glu212. The catalytic nucleophile was identified by site-directed mutagenesis in harness with the kinetics of 2,4-dintrophenyl-beta-mannobioside hydrolysis which, although very slow was associated with a dramatic decrease in KM <cite>{Bolam, 1996 #7}<cite>. The identity of the catalytic nucleophile was also revealed through site-directed mutagenesis {Bolam, 1996 #7} and its function was visualized by X-ray crystallography in which it was bound to 2-deoxy-2-fluoromannose in the acid-base mutant {Ducros, 2002 #45}. In Clan GHA, of which GH26 is a member, the residue immediately preceding the acid base in sequence is an asparagine that makes pivotal interactions with the 2-hydroxyl of the substrate. In GH26 the equivalent amino acid is a histidine, His211 in CjMan26A, although its function is conserved; it also makes important interactions with the 2-hydroxyl of the substrate {Ducros, 2002 #45}. | ||
== Three-dimensional structures == | == Three-dimensional structures == | ||
Revision as of 11:40, 29 June 2009
Substrate specificities
This family consists primarily of endo-beta1,4-mannanases, although a recent exo-acting beta- mannanase has been described. The family also contains enzymes that display beta-1,3:1,4-glucanase [1] and beta-1,3 xylanase activities.
Kinetics and Mechanism
Family GH26 enzymes are “retainers”, as shown by NMR and follow a classical Koshland double-displacement mechanism. Pre-steady state kinetics using activated substrates revealed the two phases of the reaction; the rapid initial glycosylation step (only with good leaving groups) followed by the slower deglycosylation. It should be noted that the use of substrates with a good leaving group result in a very low apparent KM, particularly with the acid-base mutant. This does not reflect tight affinity but simply that the glycosylation step (k2) is much quicker than the deglycosylation step (k3) {Bolam, 1996 #7}.
== Catalytic Residues ==\ Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4
The catalytic residues were first identified in the endo-beta1,4-mannanase CjMan26A. The catalytic acid-base is the glutamate Glu320, which is separated in sequence by ~100 residues from the catalytic nucleophile, Glu212. The catalytic nucleophile was identified by site-directed mutagenesis in harness with the kinetics of 2,4-dintrophenyl-beta-mannobioside hydrolysis which, although very slow was associated with a dramatic decrease in KM [1, 2, 2, 3, 3, 4, 4, 5, 5, 6, 7, 8, 8, 8, 8, 9, 9, 9, 9, 9, 9, 9, 9, 9, 10, 11, 12, 12, 12, 13, 13, 14, 15, 16, 17, 18, 19, 19, 20, 20, 21, 22, 23, 24, 25, 25, 25, 25, 26, 26, 27, 27, 28, 29, 30, 31, 32, 33, 33, 34, 34, 35, 35, 36, 36, 37, 38, 39, 39, 40, 40, 40, 40, 41, 41, 41, 42, 43, 44, 45, 46, 46, 47, 48, 49, 50, 51, 52, 52, 53, 54, 54, 55, 55, 55, 56, 56, 57, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78].
- First catalytic nucleophile identification
- First general acid/base residue identification
- First 3-D structure
References
- Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n |
- Araki T, Hashikawa S, and Morishita T. (2000). Cloning, sequencing, and expression in Escherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium, Vibrio sp. strain XY-214. Appl Environ Microbiol. 2000;66(4):1741-3. DOI:10.1128/AEM.66.4.1741-1743.2000 |