1MTO

Crystal structure of a Phosphofructokinase mutant from Bacillus stearothermophilus bound with fructose-6-phosphate


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.20 Å
  • R-Value Free: 0.280 
  • R-Value Work: 0.180 
  • R-Value Observed: 0.189 

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This is version 1.5 of the entry. See complete history


Literature

Reversible Ligand-Induced Dissociation of a Tryptophan-Shift Mutant of Phosphofructokinase from Bacillus stearothermophilus

Riley-Lovingshimer, M.R.Ronning, D.R.Sacchettini, J.C.Reinhart, G.D.

(2002) Biochemistry 41: 12967-12974

  • DOI: https://doi.org/10.1021/bi0263412
  • Primary Citation of Related Structures:  
    1MTO

  • PubMed Abstract: 

    The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.


  • Organizational Affiliation

    Department of Biochemistry and Biophysics and Center for Advanced Biomolecular Research, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
6-phosphofructokinase
A, B, C, D, E
A, B, C, D, E, F, G, H
319Geobacillus stearothermophilusMutation(s): 2 
Gene Names: pfk
EC: 2.7.1.11
UniProt
Find proteins for P00512 (Geobacillus stearothermophilus)
Explore P00512 
Go to UniProtKB:  P00512
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00512
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.20 Å
  • R-Value Free: 0.280 
  • R-Value Work: 0.180 
  • R-Value Observed: 0.189 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 110.682α = 90
b = 106.872β = 113.98
c = 119.588γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-12-31
    Type: Initial release
  • Version 1.1: 2008-04-28
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Data collection, Derived calculations, Structure summary
  • Version 1.4: 2021-10-27
    Changes: Database references, Structure summary
  • Version 1.5: 2024-02-14
    Changes: Data collection, Refinement description