Beta-amylase

 

Beta-amylase (alpha-1,4-glucan maltohydrolase; EC 3.2.1.2) catalyses the release of beta-anomeric maltose from the non-reducing ends of starch. It is known to function by multiple attack, several maltose molecules being produced before the maltooligosaccharide chain is released from the active site. Although known to bind a calcium ion, the metal is not involved in catalysis.

On the basis of its sequence alignment, beta-amylase has been classified as belonging to the glycoside hydrolase family 14 (GH14). It is an inverting glycoside hydrolase that is distributed throughout higher plants, oomycetes and bacteria. However, on the basis of sequence comparisons, plant and bacterial beta-amylases can be readily distinguished from each other.

 

Reference Protein and Structure

Sequence
P10538 UniProt (3.2.1.2) IPR001371 (Sequence Homologues) (PDB Homologues)
Biological species
Glycine max (Soybean) Uniprot
PDB
1bya - CRYSTAL STRUCTURES OF SOYBEAN BETA-AMYLASE REACTED WITH BETA-MALTOSE AND MALTAL: ACTIVE SITE COMPONENTS AND THEIR APPARENT ROLE IN CATALYSIS (2.2 Å) PDBe PDBsum 1bya
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1bya)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.2)

alpha-maltotriose
CHEBI:27931ChEBI
+
water
CHEBI:15377ChEBI
→
alpha-D-glucose
CHEBI:17925ChEBI
+
maltose
CHEBI:17306ChEBI
Alternative enzyme names: Beta amylase, Beta-amylase, Glycogenase, Saccharogen amylase, 1,4-alpha-D-glucan maltohydrolase,

Enzyme Mechanism

Introduction

Glu383 acts as a general base, whilst Glu186 acts as a general acid. At the first step, the general acid, Glu1186, protonates the leaving oxygen atom of the glycosidic linkage, resulting in the formation of carbonium ion intermediate. At the next stage, the general base, Glu380, abstracts the proton from a nearby water molecule to produce an activated hydroxide ion, which makes a nucleophilic attack on the carbonium ion intermediate to produce beta-maltose.

Catalytic Residues Roles

UniProt PDB* (1bya)
Asp102 Asp101A Exact role unclear, but likely to be involved in stabilising the intermediates/transition states. electrostatic stabiliser
Glu187, Glu381 Glu186A, Glu380A Acts as a general acid/base. proton shuttle (general acid/base)
Thr343 Thr342A Thr342 may stabilise the deprotonated form of Glu186, electrostatic stabiliser
Leu384 Leu383A Involved in ensuring the substrate is in the correct orientation. steric role
*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. Miyake H et al. (2002), J Biochem, 131, 587-591. Catalytic mechanism of beta-amylase from Bacillus cereus var. mycoides: chemical rescue of hydrolytic activity for a catalytic site mutant (Glu367-->Ala) by azide. PMID:11926997.
  2. Srivastava G et al. (2014), Plant Physiol Biochem, 83, 217-224. Identification of active site residues of Fenugreek β-amylase: Chemical modification and in silico approach. DOI:10.1016/j.plaphy.2014.08.005. PMID:25179433.
  3. Fazekas E et al. (2013), Biochim Biophys Acta, 1834, 1976-1981. Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates. DOI:10.1016/j.bbapap.2013.06.017. PMID:23831155.
  4. Mori T et al. (2012), J Mol Catal B Enzym, 82, 121-126. Kinetic monitoring of site-directed mutational β-amylase catalysis on a 27-MHz QCM. DOI:10.1016/j.molcatb.2012.05.019.
  5. Rejzek M et al. (2011), Mol Biosyst, 7, 718-730. Chemical genetics and cereal starch metabolism: structural basis of the non-covalent and covalent inhibition of barley β-amylase. DOI:10.1039/c0mb00204f. PMID:21085740.
  6. Ishikawa K et al. (2007), Biochemistry, 46, 792-798. Kinetic and Structural Analysis of Enzyme Sliding on a Substrate:  Multiple Attack in β-Amylase‡. DOI:10.1021/bi061605w. PMID:17223700.
  7. Kang YN et al. (2005), Biochemistry, 44, 5106-5116. Structural Analysis of Threonine 342 Mutants of Soybean β-Amylase:  Role of a Conformational Change of the Inner Loop in the Catalytic Mechanism†,‡. DOI:10.1021/bi0476580. PMID:15794648.
  8. Kang YN et al. (2004), J Mol Biol, 339, 1129-1140. The Roles of Glu186 and Glu380 in the Catalytic Reaction of Soybean β-Amylase. DOI:10.1016/j.jmb.2004.04.029. PMID:15178253.
  9. Laederach A et al. (1999), Proteins, 37, 166-175. Automated docking of maltose, 2-deoxymaltose, and maltotetraose into the soybean beta-amylase active site. PMID:10584063.
  10. Totsuka A et al. (1996), Eur J Biochem, 240, 655-659. Functional Analysis of Glu380 and Leu383 of Soybean beta-Amylase. A Proposed Action Mechanism. DOI:10.1111/j.1432-1033.1996.0655h.x.
  11. Davies G et al. (1995), Structure, 3, 853-859. Structures and mechanisms of glycosyl hydrolases. DOI:10.1016/s0969-2126(01)00220-9. PMID:8535779.
  12. TOTSUKA A et al. (1994), Eur J Biochem, 221, 649-654. Residues essential for catalytic activity of soybean beta-amylase. DOI:10.1111/j.1432-1033.1994.tb18777.x.
  13. Mikami B et al. (1994), Biochemistry, 33, 7779-7787. Crystal structures of soybean beta-amylase reacted with beta-maltose and maltal: active site components and their apparent roles in catalysis. PMID:8011643.
  14. Mikami B et al. (1993), Biochemistry, 32, 6836-6845. The 2.0-.ANG. resolution structure of soybean .beta.-amylase complexed with .alpha.-cyclodextrin. DOI:10.1021/bi00078a006.
  15. Nitta Y et al. (1989), J Biochem, 105, 573-576. Identification of glutamic acid 186 affinity-labeled by 2,3-epoxypropyl alpha-D-glucopyranoside in soybean beta-amylase. PMID:2474529.
  16. Hehre EJ et al. (1979), J Biol Chem, 254, 5942-5950. Scope and mechanism of carbohydrase action. Hydrolytic and nonhydrolytic actions of beta-amylase on alpha- and beta-maltosyl fluoride. PMID:156183.

Step 1.

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Catalytic Residues Roles

Residue Roles
Glu186A proton shuttle (general acid/base)
Glu380A proton shuttle (general acid/base)
Leu383A steric role
Thr342A electrostatic stabiliser
Asp101A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Gemma L. Holliday, Nozomi Nagano, Craig Porter