PDBsum entry 3bak

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Hydrolase PDB id
Protein chain
496 a.a. *
Waters ×269
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: N298s mutant of human pancreatic alpha-amylase in complex wi
Structure: Pancreatic alpha-amylase. Chain: a. Synonym: pa, 1,4-alpha-d- glucan glucanohydrolase. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: amy2a. Expressed in: pichia pastoris. Expression_system_taxid: 4922
1.90Å     R-factor:   0.174     R-free:   0.206
Authors: J.R.Fredriksen,R.Maurus,G.D.Brayer
Key ref: R.Maurus et al. (2008). Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity. Biochemistry, 47, 3332-3344. PubMed id: 18284212 DOI: 10.1021/bi701652t
08-Nov-07     Release date:   25-Mar-08    
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Protein chain
Pfam   ArchSchema ?
P04746  (AMYP_HUMAN) -  Pancreatic alpha-amylase
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   5 terms 
  Biochemical function     catalytic activity     8 terms  


DOI no: 10.1021/bi701652t Biochemistry 47:3332-3344 (2008)
PubMed id: 18284212  
Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity.
R.Maurus, A.Begum, L.K.Williams, J.R.Fredriksen, R.Zhang, S.G.Withers, G.D.Brayer.
A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product. In contrast, while nitrite considerably boosts the relative activity of human pancreatic alpha-amylase, its presence leads to changes in the electrostatic environment and active site conformations that substantially modify catalytic parameters and produce a novel acarbose rearrangement product. In particular, nitrite-substituted human pancreatic alpha-amylase demonstrates the unique ability to cleave acarbose into its acarviosine and maltose parts and carry out a previously unseen product elongation. In a completely unexpected turn of events, structural studies show that in azide-bound human pancreatic alpha-amylase, the normally resident chloride ion is retained in its binding site and an azide anion is found bound in an embedded side pocket in the substrate binding cleft. These results clearly indicate that azide enzymatic activation occurs via a mechanism distinct from that of the nitrite and nitrate anions.

Literature references that cite this PDB file's key reference

  PubMed id Reference
18491409 J.A.DeCaro (2008).
Methodological considerations in the use of salivary alpha-amylase as a stress marker in field research.
  Am J Hum Biol, 20, 617-619.  
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