PDBsum entry 1ix6

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Transferase PDB id
Jmol PyMol
Protein chain
396 a.a. *
Waters ×84
* Residue conservation analysis
PDB id:
Name: Transferase
Title: Aspartate aminotransferase active site mutant v39f
Structure: Aspartate aminotransferase. Chain: a. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Dimer (from PDB file)
2.20Å     R-factor:   0.205     R-free:   0.258
Authors: H.Hayashi,H.Mizuguchi,I.Miyahara,Y.Nakajima,K.Hirotsu,H.Kaga
Key ref:
H.Hayashi et al. (2003). Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis. J Biol Chem, 278, 9481-9488. PubMed id: 12488449 DOI: 10.1074/jbc.M209235200
14-Jun-02     Release date:   03-Jul-02    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00509  (AAT_ECOLI) -  Aspartate aminotransferase
396 a.a.
396 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - Aspartate transaminase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate
+ 2-oxoglutarate
= oxaloacetate
+ L-glutamate
      Cofactor: Pyridoxal 5'-phosphate
Pyridoxal 5'-phosphate
Bound ligand (Het Group name = PLP) matches with 93.75% similarity
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   2 terms 
  Biological process     biosynthetic process   4 terms 
  Biochemical function     transferase activity     7 terms  


DOI no: 10.1074/jbc.M209235200 J Biol Chem 278:9481-9488 (2003)
PubMed id: 12488449  
Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis.
H.Hayashi, H.Mizuguchi, I.Miyahara, Y.Nakajima, K.Hirotsu, H.Kagamiyama.
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.
  Selected figure(s)  
Figure 1.
Fig. 1. pH dependence of the apparent molar extinction coefficients at 430 nm of AspATs at 298 K in the presence of 50 mM buffer component(s) and 0.1 M KCl. circle , WT; , V39F; , N194A; , V39F/N194A; , V39F in the presence of a saturating concentration of maleate. The theoretical lines are drawn using Equation 3. The [E] value is set to zero, because the unprotonated aldimine has no absorbance over 400 nm (13).
Figure 5.
Fig. 5. a, schematic representation of the proposed structures of E[L]H+·S and E[L]·SH+, showing the hydrogen-bonding pattern. Lone pair electrons are expressed by shaded lobes. b, free energy levels of the reaction intermediates in the reaction pathway starting from the PLP form of AspAT and aspartate to the transition state of the 1,3-prototropic shift. Horizontal bars indicate the free-energy levels expressed in kJ·mol 1. The labels attached to the bars are defined as shown in Scheme I. The horizontal coordinate shows the chemical species sorted by the protonation state. Perpendicular to this one is the reaction coordinate. The broken bars are those of V39F. The energy levels of the transition state are adjusted between WT and V39F. The energy levels are calculated based on the data obtained in this study and Ref. 14, corrected for the difference in the pK[a] value of the amino acid -amino group (10.6 for MeAsp and 9.6 for aspartate). The rate of the 1,3-prototropic shift of WT is estimated to be 3500 s 1, based on the consideration that this step is 16% rate-determining (calculated using the equation (Dk[cat] 1)/Dk[+3] 1); see Ref. 28) in the half-reaction.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 9481-9488) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19768502 M.A.Söderberg, and N.P.Cianciotto (2010).
Mediators of lipid A modification, RNA degradation, and central intermediary metabolism facilitate the growth of Legionella pneumophila at low temperatures.
  Curr Microbiol, 60, 59-65.  
18831049 T.Tomita, T.Miyagawa, T.Miyazaki, S.Fushinobu, T.Kuzuyama, and M.Nishiyama (2009).
Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus.
  Proteins, 75, 348-359.
PDB codes: 2zp7 3cbf
17444518 R.Sathyapriya, and S.Vishveshwara (2007).
Structure networks of E. coli glutaminyl-tRNA synthetase: effects of ligand binding.
  Proteins, 68, 541-550.  
14622303 C.Rodríguez-Caso, D.Rodríguez-Agudo, A.A.Moya-García, I.Fajardo, M.A.Medina, V.Subramaniam, and F.Sánchez-Jiménez (2003).
Local changes in the catalytic site of mammalian histidine decarboxylase can affect its global conformation and stability.
  Eur J Biochem, 270, 4376-4387.  
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