5C66

E. Coli Alkaline Phosphatase in complex with tungstate


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.03 Å
  • R-Value Free: 0.240 
  • R-Value Work: 0.195 
  • R-Value Observed: 0.197 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase.

Peck, A.Sunden, F.Andrews, L.D.Pande, V.S.Herschlag, D.

(2016) J Mol Biol 428: 2758-2768

  • DOI: https://doi.org/10.1016/j.jmb.2016.05.007
  • Primary Citation of Related Structures:  
    5C66

  • PubMed Abstract: 

    The catalytic mechanisms underlying Escherichia coli alkaline phosphatase's (AP) remarkable rate enhancement have been probed extensively. Past work indicated that whereas the serine nucleophile (Ser102) electrostatically repels the product phosphate, another oxyanion, tungstate, binds more strongly in the presence of Ser102. These results predict a covalent bond between the serine nucleophile and tungstate, a model that we test herein. The crystal structure of tungstate-bound alkaline phosphatase provides evidence for a covalent adduct model and further shows that the ligand adopts trigonal bipyramidal geometry, which is infrequently observed for tungstate in small molecules and other active sites but mirrors the geometry of the presumed phosphoryl transfer transition state. The AP active site is known to stabilize another oxyanion, vanadate, in trigonal bipyramidal geometry, but the extent to which binding of either ligand reproduces the energetics of the transition state cannot be deduced from structural inspection alone. To test for transition state analog behavior, we determined the relationship between catalytic activity and affinity for tungstate and vanadate for a series of 20 AP variants. Affinity and activity were highly correlated for tungstate (r(2) = 0.89) but not vanadate (r(2) = 0.23), indicating that the tungstate•AP complex may better mimic this enzyme's transition state properties. The results herein suggest that tungstate will be a valuable tool for further dissecting AP catalysis and may prove helpful in mechanistic studies of other phosphoryl transfer enzymes.


  • Organizational Affiliation

    Department of Biochemistry, Stanford University, Beckman Center B400, Stanford, CA, 94305, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Alkaline phosphatase
A, B
459Escherichia coli K-12Mutation(s): 0 
Gene Names: phoAb0383JW0374
EC: 3.1.3.1
UniProt
Find proteins for P00634 (Escherichia coli (strain K12))
Explore P00634 
Go to UniProtKB:  P00634
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00634
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

Unit Cell:
Length ( Å )Angle ( ˚ )
a = 161.26α = 90
b = 161.26β = 90
c = 139.432γ = 120
Software Package:
Software NamePurpose
iMOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
REFMACrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2016-06-01
    Type: Initial release
  • Version 1.1: 2016-06-22
    Changes: Database references
  • Version 1.2: 2023-09-27
    Changes: Data collection, Database references, Derived calculations, Refinement description