PDBsum entry 1jxj

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Hydrolase PDB id
Protein chain
496 a.a. *
Waters ×294
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Role of mobile loop in the mechanism of human salivary amylase
Structure: Alpha-amylase, salivary. Chain: a. Synonym: salivary alpha-amyalse. 1,4-alpha-d-glucan glucanohydrolase. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Organ: salivary glands. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108
1.99Å     R-factor:   0.166     R-free:   0.198
Authors: N.Ramasubbu,C.Ragunath,Z.Wang,P.J.Mishra,L.M.Thomas
Key ref:
N.Ramasubbu et al. (2004). Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity. Eur J Biochem, 271, 2517-2529. PubMed id: 15182367 DOI: 10.1111/j.1432-1033.2004.04182.x
07-Sep-01     Release date:   14-Sep-01    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P04745  (AMY1_HUMAN) -  Alpha-amylase 1
511 a.a.
496 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Alpha-amylase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   3 terms 
  Biological process     metabolic process   3 terms 
  Biochemical function     catalytic activity     7 terms  


DOI no: 10.1111/j.1432-1033.2004.04182.x Eur J Biochem 271:2517-2529 (2004)
PubMed id: 15182367  
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.
N.Ramasubbu, C.Ragunath, P.J.Mishra, L.M.Thomas, G.Gyémánt, L.Kandra.
The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.
  Selected figure(s)  
Figure 5.
Fig. 5. Difference electron density maps(omit maps) in the mutants W58L and W58A/acarbose complex.(A) Stereodrawing of the 2Fo-Fc omit maps corresponding to residues 58 and 59 in the mutant W58L. (B) Stereodrawing of the 2Fo-Fc omitting density maps corresponding to the bound oligosaccharide in W58A. This complex is made up of a trisaccharide and is named according to subsite location. The electron density map has been contoured at 1 and the final refined coordinates of the corresponding oligosaccharide residues are overlaid.
Figure 7.
Fig. 7. Comparison of the conformation of the bound pseudooligosaccharide in human and fungal -amylases. Superposition of the glycone subsites in HSAmy (thick lines; PDB Code 1mfv) and TAKA-amylase (thin lines; PDB Code 7taa) along with the bound pseudooligosaccharide. These two structures were superposed by fitting the spatial location of the three catalytic residues (equivalent to Asp197, Glu233 and Asp300 in HSAmy). The two bound pseudooligosaccharides (HSAmy vs. TAKA-amylase) traverse two different paths beyond the subsite –2 as shown by the dotted spheres. Note that Trp58 and Trp59 of HSAmy would encounter severe steric interaction if the bound oligosaccharide in HSAmy adopted a conformation as in TAKA-amylase.
  The above figures are reprinted by permission from the Federation of European Biochemical Societies: Eur J Biochem (2004, 271, 2517-2529) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21426903 L.C.Tsai, C.H.Hsiao, W.Y.Liu, L.M.Yin, and L.F.Shyur (2011).
Structural basis for the inhibition of 1,3-1,4-β-d-glucanase by noncompetitive calcium ion and competitive Tris inhibitors.
  Biochem Biophys Res Commun, 407, 593-598.  
20857221 M.Najafian, A.Ebrahim-Habibi, N.Hezareh, P.Yaghmaei, K.Parivar, and B.Larijani (2011).
Trans-chalcone: a novel small molecule inhibitor of mammalian alpha-amylase.
  Mol Biol Rep, 38, 1617-1620.  
18951906 C.Ragunath, S.G.Manuel, V.Venkataraman, H.B.Sait, C.Kasinathan, and N.Ramasubbu (2008).
Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding.
  J Mol Biol, 384, 1232-1248.  
18601990 M.R.Loizzo, A.M.Saab, R.Tundis, F.Menichini, M.Bonesi, V.Piccolo, G.A.Statti, Cindio, P.J.Houghton, and F.Menichini (2008).
In vitro inhibitory activities of plants used in Lebanon traditional medicine against angiotensin converting enzyme (ACE) and digestive enzymes related to diabetes.
  J Ethnopharmacol, 119, 109-116.  
17949435 S.G.Manuel, C.Ragunath, H.B.Sait, E.A.Izano, J.B.Kaplan, and N.Ramasubbu (2007).
Role of active-site residues of dispersin B, a biofilm-releasing beta-hexosaminidase from a periodontal pathogen, in substrate hydrolysis.
  FEBS J, 274, 5987-5999.  
16835869 H.M.Rawel, S.K.Frey, K.Meidtner, J.Kroll, and F.J.Schweigert (2006).
Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.
  Mol Nutr Food Res, 50, 705-713.  
15981193 I.Damager, M.T.Jensen, C.E.Olsen, A.Blennow, B.L.Møller, B.Svensson, and M.S.Motawia (2005).
Chemical synthesis of a dual branched malto-decaose: a potential substrate for alpha-amylases.
  Chembiochem, 6, 1224-1233.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time.