RCSB PDB - 3BAJ: Human Pancreatic Alpha-Amylase in Complex with Nitrate and Acarbose

 3BAJ

Human Pancreatic Alpha-Amylase in Complex with Nitrate and Acarbose


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.225 
  • R-Value Work: 0.174 

Starting Model: experimental
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Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted AREClick on this verticalbar to view detailsBest fitted NAGClick on this verticalbar to view details

This is version 2.3 of the entry. See complete history


Literature

Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity

Maurus, R.Begum, A.Williams, L.K.Fredriksen, J.R.Zhang, R.Withers, S.G.Brayer, G.D.

(2008) Biochemistry 47: 3332-3344

  • DOI: https://doi.org/10.1021/bi701652t
  • Primary Citation of Related Structures:  
    2QMK, 2QV4, 3BAI, 3BAJ, 3BAK, 3BAW, 3BAX, 3BAY

  • PubMed Abstract: 

    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.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Pancreatic alpha-amylase496Homo sapiensMutation(s): 0 
Gene Names: AMY2A
EC: 3.2.1.1
UniProt & NIH Common Fund Data Resources
Find proteins for P04746 (Homo sapiens)
Explore P04746 
Go to UniProtKB:  P04746
PHAROS:  P04746
GTEx:  ENSG00000243480 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP04746
Glycosylation
Glycosylation Sites: 1Go to GlyGen: P04746-1
Sequence Annotations
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  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.225 
  • R-Value Work: 0.174 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 53.14α = 90
b = 69.2β = 90
c = 132.66γ = 90
Software Package:
Software NamePurpose
MAR345dtbdata collection
CNSrefinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing

Structure Validation

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Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted AREClick on this verticalbar to view detailsBest fitted NAGClick on this verticalbar to view details

Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2008-03-25
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Non-polymer description, Version format compliance
  • Version 2.0: 2019-12-25
    Changes: Database references, Derived calculations, Polymer sequence
  • Version 2.1: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Data collection, Derived calculations, Structure summary
  • Version 2.2: 2023-08-30
    Changes: Data collection, Database references, Refinement description, Structure summary
  • Version 2.3: 2024-10-30
    Changes: Structure summary