Glycoside Hydrolase Family 84

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• Author: ^^^Ian Greig^^^
• Responsible Curator: ^^^David Vocadlo^^^

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Glycoside Hydrolase Family GH84
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH84.html

Substrate specificities

In contrast to the b-hexosaminidases of GH20 a relaxed specificity for substitutions of the N-acyl group is observed with residues significantly more bulky than the N-acyl group being tolerated.REF Content is to be added here.

This is an example of how to make references to a journal article [1]. (See the References section below). Multiple references can go in the same place like this [1, 2]. You can even cite books using just the ISBN [3]. References that are not in PubMed can be typed in by hand [4].

Kinetics and Mechanism

The most extensive kinetic studies have been carried out on Human O-GlcNAcase. Neighbouring group participation.[5] Substrate distortion.[6, 7] General acid catalysis operative for substrates possessing leaving groups with pKas greater than approximately XXX. For either O- or S-glycosides possessing leaving groups with pKas below XXX the leaving group will depart at the anion.[6, 8] Nuclear isoform (TRUNCATION) of Human O-GlcNAcase retains similar kinetic properties and inhibitory patterns as the cytosolic isoform consistent with hexosaminidase activity residing in the XXX domains.[9]

Catalytic Residues

Studies of two mutants of human O-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.[10]

The mutant Asp175Ala displayed marked reductions in activity (V and (V/K)) towards aryl N-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both O-aryl and S-aryl N-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards O-aryl N-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the N-acyl nucleophile.

Three-dimensional structures

The reported crystallization of Clostridium perfringens NagJ[11] was followed by solved structures for that enzyme[12] and Bacteroides thetaiotaomicron b-hexosaminidase[12]. A series of crystallographic studies on Bacteroides thetaiotaomicron b-hexosaminidase using a variety of small molecules define the conformational itinerary for this family. Substrate distortion: WT + azepane [13], WT + difluoroacetyl [14], 4C1 intermediate: WT + thiazoline [5], general acid mutants Asp243Asn + 5-fluorooxazoline derived from b-1,5-difluoroglucosaminide,[14] Asp243Asn + oxazoline derived from 4-methylumbelliferyl b-glucosaminide,[14].

Family Firsts

First sterochemistry determination

Cite some reference here, with a short (1-2 sentence) explanation [1].

First catalytic nucleophile identification

Cite some reference here, with a short (1-2 sentence) explanation [4].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation [2].

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [3].

References

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4. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006

10. Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants. Biochemistry. 2006 Mar 21;45(11):3835-44.

    Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out: DOI:10.1021/bi052370b PMID:16533067