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

GH84 contains β-N-acetylglucosaminidases and β-N-acetylhyaluronidase activities. Human O-GlcNAcase is a cytosolic enzyme whose in vivo targets are glycoprotein serine and threonine residues modified by a single β-linked GlcNAc residue. In contrast to the β-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.[1]

Kinetics and Mechanism

Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human O-GlNAcase.[1] More recent studies of this enzyme have extended such investigated variations in rates of reaction (V/K) with both nucleophile and leaving group structures.[2] For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on V/K for leaving groups with a pK_a below 11 (with the chemical step rate-determining for substrates with higher pK_a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (D_N*A_N) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a pK_a greater than approximately 7; a concerted (A_ND_N) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower pK_as (consistent with prior studies of S-glucosaminide hydrolysis[3]). Substrate distortion.[2, 4] A truncated, nuclear-localized isoform of human O-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the N-terminal domain.[5]

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.[6]

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[7] was followed by solved structures for that enzyme[8] and Bacteroides thetaiotaomicron β-hexosaminidase[8]. A series of crystallographic studies on Bacteroides thetaiotaomicron β-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. [9] This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-β-D-glucopyranoside.[10] Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1 conformation.[1] Subsequent studies have shown that oxazoline intermediates are also bound to the general acid mutant Asp243Asn + 5-fluorooxazoline derived from b-1,5-difluoroglucosaminide,[10] Asp243Asn + oxazoline derived from 4-methylumbelliferyl b-glucosaminide,[10].

Family Firsts

First sterochemistry determination

¹H-NMR studies of human O-GlcNAcase established that the β-configured hemiacetal product is formed prior to anomerisation.[2].

First catalytic nucleophile identification

This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.[1].

First general acid/base residue identification

Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.[6].

First 3-D structure

The structures of Bacteroides thetaiotaomicron O-GlcNAcase[11] and Clostridium perfringens NagJ[8].

References

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1. Macauley MS, Whitworth GE, Debowski AW, Chin D, and Vocadlo DJ. (2005). O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem. 2005;280(27):25313-22. DOI:10.1074/jbc.M413819200 |

6. 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

12. 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 |
13. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 |
14. Robert V. Stick and Spencer J. Williams. (2009) Carbohydrates. Elsevier Science.

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