Beta-N-acetylhexosaminidase

 

Chitin, the second most abundant polysaccharide on earth, is degraded by chitinases and chitobiases. This entry represents the glycosidase family 20 chitobiase which digests the beta-1,4-glycosidic bonds in N-acetylglucosamine (GlcNAc) oligomers (mainly dimers). It has a retaining mechanism. Human beta-hexosaminidase A

 

Reference Protein and Structure

Sequence
P06865 UniProt (3.2.1.52) IPR025705 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
2gjx - Crystallographic structure of human beta-Hexosaminidase A (2.8 Å) PDBe PDBsum 2gjx
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 2gjx)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.52)

N,N'-diacetylchitobiose
CHEBI:28681ChEBI
+
water
CHEBI:15377ChEBI
→
N-acetyl-D-glucosamine
CHEBI:506227ChEBI
Alternative enzyme names: N-acetyl-beta-D-hexosaminidase, N-acetyl-beta-hexosaminidase, N-acetylhexosaminidase, Beta-N-acetyl-D-hexosaminidase, Beta-N-acetylglucosaminidase, Beta-D-N-acetylhexosaminidase, Beta-D-hexosaminidase, Beta-acetylaminodeoxyhexosidase, Beta-acetylhexosaminidinase, Beta-hexosaminidase, Hexosaminidase, Hexosaminidase A, N-acetyl-beta-glucosaminidase,

Enzyme Mechanism

Introduction

Glu540 acts as a general acid, to protonate the glycosidic oxygen atom, the O1 atom, leading to the transition state with oxocarbenium ion character. The O7 oxygen atom of the N-acetyl group makes a nucleophilic attack on the C1 atom of the transition state. This nucleophilic substitution seems to be S1-like reaction. A water, activated by Glu540, makes a nucleophilic attack on the C1 atom of the intermediate, leading again to the transition state with oxocarbenium ion character. The covalent bond between the C1 atom and the O7 oxygen atom is broken, and finally the product is formed.

Catalytic Residues Roles

UniProt PDB* (2gjx)
Asp539 Asp539(512)A Modulates the pKa of both the catalytic glutamate, but also helps activate the N-acetyl group of the substrate, by forming a hydrogen bond with the N2 atom of the N-acetyl group. It also helps stabilise the positively charged reaction intermediates and transition states that are formed during the course of the reaction. modifies pKa, electrostatic stabiliser, steric role
Glu540 Glu540(513)A Acts as a general acid/base. proton shuttle (general acid/base)
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Prag G et al. (2000), J Mol Biol, 300, 611-617. Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540. DOI:10.1006/jmbi.2000.3906. PMID:10884356.
  2. Drouillard S et al. (1997), Biochem J, 328 ( Pt 3), 945-949. Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. PMID:9396742.
  3. Tews I et al. (1996), Nat Struct Biol, 3, 638-648. Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. PMID:8673609.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu540(513)A proton shuttle (general acid/base)
Asp539(512)A modifies pKa, electrostatic stabiliser, steric role

Chemical Components

Step 1.

Introduction

Glu323 acts as a general acid, to protonate the glyosidic oxygen atom, the O1 atom, leading to the transition state with oxocarbenium ion character. Asp322 provides a negative charge that stablise the positive charge developed on the oxazinium ion. The O7 oxygen atom of the N-acetyl group makes a nucleophilic attack on the C1 atom of the transition state. This nucleophilic substitution seems to be S1-like reaction. A water, activated by Glu323, makes a nucleophilic attack on the C1 atom of the intermediate, leading again to the transition state with oxocarbenium ion character. The covalent bond between the C1 atom and the O7 oxygen atom is broken, and finally the product is formed.

Catalytic Residues Roles

UniProt PDB* (2gjx)
Glu323 Glu323(301)A Acts as a general acid-base residue. proton acceptor, proton donor
Asp322, Glu323 Asp322(300)A, Glu323(301)A electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic substitution

References

  1. Passos Ó et al. (2011), J Phys Chem B, 115, 14751-14759. QM/MM Study of the Catalytic Mechanism of GalNAc Removal from GM2 Ganglioside Catalyzed by Human β-HexosaminidaseA. DOI:/10.1021/jp205826n.
  2. Prag G et al. (2000), J Mol Biol, 300, 611-617. Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540. DOI:10.1006/jmbi.2000.3906. PMID:10884356.
  3. Drouillard S et al. (1997), Biochem J, 328 ( Pt 3), 945-949. Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. PMID:9396742.
  4. Tews I et al. (1996), Nat Struct Biol, 3, 638-648. Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. PMID:8673609.

Step 1. The acid-base residue Glu323 protonates the oxygen of the scissile glyosidic bond oxygen.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu323(301)A proton donor
Asp322(300)A electrostatic stabiliser

Chemical Components

proton transfer

Step 2. An incoming water molecule attack the anomeric carbon and maintain the double displacement mechanism.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu323(301)A proton acceptor
Asp322(300)A electrostatic stabiliser

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. The acid-base residue Glu323 protonates the oxygen of the scissile glyosidic bond oxygen.

Step 2. An incoming water molecule attack the anomeric carbon and maintain the double displacement mechanism.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday, Yordanos Abeje