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PDBsum entry 1xh0

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Hydrolase PDB id
1xh0
Jmol
Contents
Protein chain
496 a.a. *
Ligands
NAG
AAO
Waters ×280
* Residue conservation analysis
PDB id:
1xh0
Name: Hydrolase
Title: Structure of the n298s variant of human pancreatic alpha-amy complexed with acarbose
Structure: Alpha-amylase, pancreatic. Chain: a. Synonym: 1,4-alpha-d-glucan glucanohydrolase, pancreatic al amylase, pa. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: pichia pastoris. Expression_system_taxid: 4922
Resolution:
2.00Å     R-factor:   0.164     R-free:   0.205
Authors: R.Maurus,A.Begum,H.H.Kuo,A.Racaza,S.Numao,C.M.Overall,S.G.Wi G.D.Brayer
Key ref:
R.Maurus et al. (2005). Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase. Protein Sci, 14, 743-755. PubMed id: 15722449 DOI: 10.1110/ps.041079305
Date:
17-Sep-04     Release date:   24-May-05    
PROCHECK
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 Headers
 References

Protein chain
Pfam   ArchSchema ?
P04746  (AMYP_HUMAN) -  Pancreatic alpha-amylase
Seq:
Struc:
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.3.2.1.1  - 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   5 terms 
  Biochemical function     catalytic activity     8 terms  

 

 
DOI no: 10.1110/ps.041079305 Protein Sci 14:743-755 (2005)
PubMed id: 15722449  
 
 
Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
R.Maurus, A.Begum, H.H.Kuo, A.Racaza, S.Numao, C.Andersen, J.W.Tams, J.Vind, C.M.Overall, S.G.Withers, G.D.Brayer.
 
  ABSTRACT  
 
The mechanism of allosteric activation of alpha-amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride-dependent N298S variant of human pancreatic alpha-amylase (HPA) and a chloride-independent TAKA-amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH-dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride-dependent alpha-amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride-independent alpha-amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride-dependence into the chloride-independent TAKA-amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride-dependent alpha-amylases.
 
  Selected figure(s)  
 
Figure 5.
Figure 5. Schematic representations of the hydrogen bond interactions formed within the chloride binding sites in wild-type HPA (and with bound acarbose) (A), N298S HPA with bound chloride (B), N298S HPA with both bound chloride and acarbose (C), N298S HPA in the absence of chloride (D), and N298S HPA in the absence of chloride, with acarbose bound (E). Note that in the N298S structure without bound chloride, the side chains of D300 and E233 are substantially shifted. For the N298S HPA-acarbose complex (in the absence of chloride), the side chain of E233 takes on two orientations that are approximately equally populated. Here conformation 1 is that found in the wild-type enzyme, while conformation 2 is that observed for the N298S variant without bound chloride and in the absence of acarbose. Bound water molecules are indicated by "Wat" and hydrogen bonds are designated by dashed lines.
Figure 6.
Figure 6. A schematic diagram summarizing the hydrogen bond interactions (dashed lines) to acarbose, when this inhibitor is bound in the active site of the N298S variant of HPA in the presence (A) and the absence (B) of chloride ion. The normal product of enzymatic inhibitor rearrangement is found in the presence of chloride. Surprisingly, an extended inhibitor product, with an additional glucose unit in binding subsite +3, is found in the absence of chloride (indicated by the dashed box). Active site cleft subsite binding locations have been indicated adjacent to each inhibitor sugar ring. Other prominent hydrogen bonding differences between these complexes occur in subsites -3 and +2. The water molecule believed to play a role in nucleophilic attack on the covalent intermediate generated during hydrolysis of substrates, has been indicated with an *.
 
  The above figures are reprinted by permission from the Protein Society: Protein Sci (2005, 14, 743-755) copyright 2005.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21397496 S.Park, S.Hyun, and J.Yu (2011).
Selective α-glucosidase substrates and inhibitors containing short aromatic peptidyl moieties.
  Bioorg Med Chem Lett, 21, 2441-2444.  
19476481 J.Pytelková, J.Hubert, M.Lepsík, J.Sobotník, R.Sindelka, I.Krízková, M.Horn, and M.Mares (2009).
Digestive alpha-amylases of the flour moth Ephestia kuehniella--adaptation to alkaline environment and plant inhibitors.
  FEBS J, 276, 3531-3546.  
19329633 L.J.Gourlay, I.Santi, A.Pezzicoli, G.Grandi, M.Soriani, and M.Bolognesi (2009).
Group B streptococcus pullulanase crystal structures in the context of a novel strategy for vaccine development.
  J Bacteriol, 191, 3544-3552.
PDB codes: 3faw 3fax
18331662 W.C.Obiro, T.Zhang, and B.Jiang (2008).
The nutraceutical role of the Phaseolus vulgaris alpha-amylase inhibitor.
  Br J Nutr, 100, 1.  
17592362 R.Quezada-Calvillo, C.C.Robayo-Torres, Z.Ao, B.R.Hamaker, A.Quaroni, G.D.Brayer, E.E.Sterchi, S.S.Baker, and B.L.Nichols (2007).
Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose.
  J Pediatr Gastroenterol Nutr, 45, 32-43.  
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. Where a reference describes a PDB structure, the PDB codes are shown on the right.