1B4S

STRUCTURE OF NUCLEOSIDE DIPHOSPHATE KINASE H122G MUTANT


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.305 
  • R-Value Work: 0.203 
  • R-Value Observed: 0.203 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase.

Admiraal, S.J.Schneider, B.Meyer, P.Janin, J.Veron, M.Deville-Bonne, D.Herschlag, D.

(1999) Biochemistry 38: 4701-4711

  • DOI: https://doi.org/10.1021/bi9827565
  • Primary Citation of Related Structures:  
    1B4S

  • PubMed Abstract: 

    The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.


  • Organizational Affiliation

    Department of Biochemistry, Beckman Center B400, Stanford University, Stanford, California 94305-5307, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
NUCLEOSIDE DIPHOSPHATE KINASE
A, B, C
155Dictyostelium discoideumMutation(s): 1 
EC: 2.7.4.6
UniProt
Find proteins for P22887 (Dictyostelium discoideum)
Explore P22887 
Go to UniProtKB:  P22887
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP22887
Sequence Annotations
Expand
  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.305 
  • R-Value Work: 0.203 
  • R-Value Observed: 0.203 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 70α = 90
b = 70β = 90
c = 151.77γ = 120
Software Package:
Software NamePurpose
X-PLORmodel building
X-PLORrefinement
DENZOdata reduction
SCALEPACKdata scaling
X-PLORphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1999-06-15
    Type: Initial release
  • Version 1.1: 2008-03-24
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-11-03
    Changes: Database references, Derived calculations, Other
  • Version 1.4: 2023-08-02
    Changes: Refinement description