1IX8

Aspartate Aminotransferase Active Site Mutant V39F/N194A


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.259 
  • R-Value Work: 0.202 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis

Hayashi, H.Mizuguchi, H.Miyahara, I.Nakajima, Y.Hirotsu, K.Kagamiyama, H.

(2003) J Biol Chem 278: 9481-9488

  • DOI: https://doi.org/10.1074/jbc.M209235200
  • Primary Citation of Related Structures:  
    1IX6, 1IX7, 1IX8

  • PubMed Abstract: 

    Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.


  • Organizational Affiliation

    Department of Biochemistry, Osaka Medical College, Takatsuki 569-8686, Japan.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Aspartate Aminotransferase396Escherichia coliMutation(s): 2 
EC: 2.6.1.1
UniProt
Find proteins for P00509 (Escherichia coli (strain K12))
Explore P00509 
Go to UniProtKB:  P00509
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00509
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
PLP
Query on PLP

Download Ideal Coordinates CCD File 
B [auth A]PYRIDOXAL-5'-PHOSPHATE
C8 H10 N O6 P
NGVDGCNFYWLIFO-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.259 
  • R-Value Work: 0.202 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 154.973α = 90
b = 88.739β = 90
c = 80.298γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
X-PLORmodel building
X-PLORrefinement
X-PLORphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-07-03
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-12-27
    Changes: Data collection