PDBsum entry 1cpu

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Hydrolase PDB id
Protein chain
496 a.a. *
Waters ×261
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Subsite mapping of the active site of human pancreatic alpha using substrates, the pharmacological inhibitor acarbose, a active site variant
Structure: Protein (alpha-amylase). Chain: a. Engineered: yes. Other_details: modified acarbose bound at active site
Source: Homo sapiens. Human. Organism_taxid: 9606. Organ: pancreas. Expressed in: pichia pastoris. Expression_system_taxid: 4922
2.00Å     R-factor:   0.170    
Authors: G.D.Brayer,G.Sidhu,R.Maurus,E.H.Rydberg,C.Braun,Y.Wang,N.T.N C.M.Overall,S.G.Withers
Key ref:
G.D.Brayer et al. (2000). Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry, 39, 4778-4791. PubMed id: 10769135 DOI: 10.1021/bi9921182
07-Jun-99     Release date:   14-Jun-99    
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/bi9921182 Biochemistry 39:4778-4791 (2000)
PubMed id: 10769135  
Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.
G.D.Brayer, G.Sidhu, R.Maurus, E.H.Rydberg, C.Braun, Y.Wang, N.T.Nguyen, C.M.Overall, S.G.Withers.
We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.

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
20877900 A.K.Verma, and R.Pratap (2010).
The biological potential of flavones.
  Nat Prod Rep, 27, 1571-1593.  
20461849 F.Cardona, A.Goti, C.Parmeggiani, P.Parenti, M.Forcella, P.Fusi, L.Cipolla, S.M.Roberts, G.J.Davies, and T.M.Gloster (2010).
Casuarine-6-O-alpha-D-glucoside and its analogues are tight binding inhibitors of insect and bacterial trehalases.
  Chem Commun (Camb), 46, 2629-2631.
PDB code: 2wyn
19411257 T.J.Morley, L.M.Willis, C.Whitfield, W.W.Wakarchuk, and S.G.Withers (2009).
A new sialidase mechanism: bacteriophage K1F endo-sialidase is an inverting glycosidase.
  J Biol Chem, 284, 17404-17410.  
18214874 C.A.Tarling, K.Woods, R.Zhang, H.C.Brastianos, G.D.Brayer, R.J.Andersen, and S.G.Withers (2008).
The search for novel human pancreatic alpha-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts.
  Chembiochem, 9, 433-438.  
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.  
18613721 S.Cheluvaraja, M.Mihailescu, and H.Meirovitch (2008).
Entropy and free energy of a mobile protein loop in explicit water.
  J Phys Chem B, 112, 9512-9522.  
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.  
17901056 T.Jank, T.Giesemann, and K.Aktories (2007).
Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding.
  J Biol Chem, 282, 35222-35231.  
17199626 F.Barni, A.Berti, C.Rapone, and G.Lago (2006).
Alpha-amylase kinetic test in bodily single and mixed stains.
  J Forensic Sci, 51, 1389-1396.  
15785807 M.Egido-Gabás, P.Serrano, J.Casas, A.Llebaria, and A.Delgado (2005).
New aminocyclitols as modulators of glucosylceramide metabolism.
  Org Biomol Chem, 3, 1195-1201.  
15722449 R.Maurus, A.Begum, H.H.Kuo, A.Racaza, S.Numao, C.Andersen, J.W.Tams, J.Vind, C.M.Overall, S.G.Withers, and G.D.Brayer (2005).
Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
  Protein Sci, 14, 743-755.
PDB codes: 1xgz 1xh0 1xh1 1xh2
16030022 X.Robert, R.Haser, H.Mori, B.Svensson, and N.Aghajari (2005).
Oligosaccharide binding to barley alpha-amylase 1.
  J Biol Chem, 280, 32968-32978.
PDB codes: 1rp8 1rp9 1rpk
14660599 K.S.Bak-Jensen, G.André, T.E.Gottschalk, G.Paës, V.Tran, and B.Svensson (2004).
Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1.
  J Biol Chem, 279, 10093-10102.  
15182367 N.Ramasubbu, C.Ragunath, P.J.Mishra, L.M.Thomas, G.Gyémánt, and L.Kandra (2004).
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.
  Eur J Biochem, 271, 2517-2529.
PDB codes: 1jxj 1nm9
15304511 S.Numao, I.Damager, C.Li, T.M.Wrodnigg, A.Begum, C.M.Overall, G.D.Brayer, and S.G.Withers (2004).
In situ extension as an approach for identifying novel alpha-amylase inhibitors.
  J Biol Chem, 279, 48282-48291.
PDB codes: 1u2y 1u30 1u33
12482867 A.Linden, O.Mayans, W.Meyer-Klaucke, G.Antranikian, and M.Wilmanns (2003).
Differential regulation of a hyperthermophilic alpha-amylase with a novel (Ca,Zn) two-metal center by zinc.
  J Biol Chem, 278, 9875-9884.
PDB codes: 1mwo 1mxd 1mxg
14511369 N.Oudjeriouat, Y.Moreau, M.Santimone, B.Svensson, G.Marchis-Mouren, and V.Desseaux (2003).
On the mechanism of alpha-amylase.
  Eur J Biochem, 270, 3871-3879.  
12392547 G.Gyémánt, G.Hovánszki, and L.Kandra (2002).
Subsite mapping of the binding region of alpha-amylases with a computer program.
  Eur J Biochem, 269, 5157-5162.  
12423336 H.Mori, K.S.Bak-Jensen, and B.Svensson (2002).
Barley alpha-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and is critical for activity and substrate specificity.
  Eur J Biochem, 269, 5377-5390.  
12392304 K.Lorentz (2002).
An ideal substrate for the measurement of pancreatic amylase?
  Clin Chem Lab Med, 40, 781-785.  
12021442 N.Aghajari, G.Feller, C.Gerday, and R.Haser (2002).
Structural basis of alpha-amylase activation by chloride.
  Protein Sci, 11, 1435-1441.
PDB codes: 1jd7 1jd9 1l0p
11291107 I.Warshawsky, and G.L.Hortin (2001).
Effect of substrate size on immunoinhibition of amylase activity.
  J Clin Lab Anal, 15, 64-70.  
11128586 L.Kandra, and G.Gyémánt (2000).
Examination of the active sites of human salivary alpha-amylase (HSA).
  Carbohydr Res, 329, 579-585.  
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.