1OS1

Structure of Phosphoenolpyruvate Carboxykinase complexed with ATP,Mg, Ca and pyruvate.


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.250 
  • R-Value Work: 0.200 
  • R-Value Observed: 0.204 

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This is version 2.0 of the entry. See complete history


Literature

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin.

Sudom, A.Walters, R.Pastushok, L.Goldie, D.Prasad, L.Delbaere, L.T.Goldie, H.

(2003) J Bacteriol 185: 4233-4242

  • DOI: https://doi.org/10.1128/JB.185.14.4233-4242.2003
  • Primary Citation of Related Structures:  
    1OS1

  • PubMed Abstract: 

    The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.


  • Organizational Affiliation

    Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Phosphoenolpyruvate carboxykinase [ATP]540Escherichia coli K-12Mutation(s): 0 
EC: 4.1.1.49
UniProt
Find proteins for P22259 (Escherichia coli (strain K12))
Explore P22259 
Go to UniProtKB:  P22259
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP22259
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.250 
  • R-Value Work: 0.200 
  • R-Value Observed: 0.204 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 126.2α = 90
b = 95.2β = 95.2
c = 46.8γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
TRUNCATEdata reduction
AMoREphasing
X-PLORrefinement
CCP4data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-09-23
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Source and taxonomy, Version format compliance
  • Version 1.3: 2023-08-16
    Changes: Data collection, Database references, Derived calculations, Refinement description
  • Version 2.0: 2023-11-15
    Changes: Atomic model, Data collection, Derived calculations