PDBsum entry 3ij9

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Hydrolase PDB id
Protein chain
496 a.a. *
B0D ×2
Waters ×307
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Directed 'in situ' elongation as a strategy to characterize the covalent glycosyl-enzyme catalytic intermediate of human pancreatic a-amylase
Structure: Pancreatic alpha-amylase. Chain: a. Fragment: human pancreatic alpha-amylase. Synonym: pa, 1,4-alpha-d-glucan glucanohydrolase. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: amy2a. Expressed in: pichia pastoris. Expression_system_taxid: 4922
1.85Å     R-factor:   0.184     R-free:   0.207
Authors: C.Li,R.Zhang,S.G Withers,G.D.Brayer
Key ref: R.Zhang et al. (2009). Directed "in situ" inhibitor elongation as a strategy to structurally characterize the covalent glycosyl-enzyme intermediate of human pancreatic alpha-amylase. Biochemistry, 48, 10752-10764. PubMed id: 19803533 DOI: 10.1021/bi901400p
04-Aug-09     Release date:   27-Oct-09    
Go to PROCHECK summary

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 1 residue position (black cross)

 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/bi901400p Biochemistry 48:10752-10764 (2009)
PubMed id: 19803533  
Directed "in situ" inhibitor elongation as a strategy to structurally characterize the covalent glycosyl-enzyme intermediate of human pancreatic alpha-amylase.
R.Zhang, C.Li, L.K.Williams, B.P.Rempel, G.D.Brayer, S.G.Withers.
While covalent catalytic intermediates of retaining alpha-transglycosylases have been structurally characterized previously, no such information for a hydrolytic alpha-amylase has been obtained. This study presents a new "in situ" enzymatic elongation methodology that, for the first time, has allowed the isolation and structural characterization of a catalytically competent covalent glycosyl-enzyme intermediate with human pancreatic alpha-amylase. This has been achieved by the use of a 5-fluoro-beta-l-idosyl fluoride "warhead" in conjunction with either alpha-maltotriosyl fluoride or 4'-O-methyl-alpha-maltosyl fluoride as elongation agents. This generates an oligosaccharyl-5-fluoroglycosyl fluoride that then reacts with the free enzyme. The resultant covalent intermediates are extremely stable, with hydrolytic half-lives on the order of 240 h for the trisaccharide complex. In the presence of maltose, however, they undergo turnover via transglycosylation according to a half-life of less than 1 h. Structural studies of intermediate complexes unambiguously show the covalent attachment of a 5-fluoro-alpha-l-idosyl moiety in the chair conformation to the side chain of the catalytic nucleophile D197. The elongated portions of the intermediate complexes are found to bind in the high-affinity -2 and -3 binding subsites, forming extensive hydrogen-bonding interactions. Comparative structural analyses with the related noncovalent complex formed by acarbose highlight the structural rigidity of the enzyme surface during catalysis and the key role that substrate conformational flexibility must play in this process. Taken together, the structural data provide atomic details of several key catalytic steps. The scope of this elongation approach to probe the active sites and catalytic mechanisms of alpha-amylases is further demonstrated through preliminary experiments with porcine pancreatic alpha-amylase.

Literature references that cite this PDB file's key reference

  PubMed id Reference
21111049 X.Qin, L.Ren, X.Yang, F.Bai, L.Wang, P.Geng, G.Bai, and Y.Shen (2011).
Structures of human pancreatic α-amylase in complex with acarviostatins: Implications for drug design against type II diabetes.
  J Struct Biol, 174, 196-202.
PDB codes: 3old 3ole 3olg 3oli
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