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PDBsum entry 2bif

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protein ligands metals Protein-protein interface(s) links
Transferase, hydrolase PDB id
2bif
Jmol
Contents
Protein chains
432 a.a. *
Ligands
BOG ×3
F6P ×2
PO4 ×3
ANP
SIN ×2
Metals
_MG
Waters ×275
* Residue conservation analysis
PDB id:
2bif
Name: Transferase, hydrolase
Title: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase h256a mutant with f6p in phosphatase active site
Structure: Protein (6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase). Chain: a, b. Engineered: yes. Mutation: yes. Other_details: bifunctional enzyme, also is ec 3.1.3.46
Source: Rattus norvegicus. Norway rat. Organism_taxid: 10116. Cell_line: bl21. Organ: testis. Gene: rt2k. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_cell_line: bl21.
Biol. unit: Dimer (from PQS)
Resolution:
2.40Å     R-factor:   0.200     R-free:   0.244
Authors: M.H.Yuen,C.A.Hasemann
Key ref:
M.H.Yuen et al. (1999). Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities. J Biol Chem, 274, 2176-2184. PubMed id: 9890980 DOI: 10.1074/jbc.274.4.2176
Date:
26-Oct-98     Release date:   16-Feb-99    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P25114  (F264_RAT) -  6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4
Seq:
Struc:
469 a.a.
432 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 4 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class 1: E.C.2.7.1.105  - 6-phosphofructo-2-kinase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP + D-fructose 6-phosphate = ADP + beta-D-fructose 2,6-bisphosphate
ATP
+
D-fructose 6-phosphate
Bound ligand (Het Group name = F6P)
corresponds exactly
=
ADP
Bound ligand (Het Group name = ANP)
matches with 81.00% similarity
+ beta-D-fructose 2,6-bisphosphate
   Enzyme class 2: E.C.3.1.3.46  - Fructose-2,6-bisphosphate 2-phosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Beta-D-fructose 2,6-bisphosphate + H2O = D-fructose 6-phosphate + phosphate
Beta-D-fructose 2,6-bisphosphate
+ H(2)O
=
D-fructose 6-phosphate
Bound ligand (Het Group name = F6P)
corresponds exactly
+
phosphate
Bound ligand (Het Group name = PO4)
corresponds exactly
Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     metabolic process   6 terms 
  Biochemical function     catalytic activity     8 terms  

 

 
    reference    
 
 
DOI no: 10.1074/jbc.274.4.2176 J Biol Chem 274:2176-2184 (1999)
PubMed id: 9890980  
 
 
Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities.
M.H.Yuen, H.Mizuguchi, Y.H.Lee, P.F.Cook, K.Uyeda, C.A.Hasemann.
 
  ABSTRACT  
 
Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.
 
  Selected figure(s)  
 
Figure 3.
Fig. 3. Model of Fru-2,6-P[2] bound to the RT2K-Wo active site. We have constructed a model of Fru-2,6-P[2] binding to the Fru-2,6-Pase active site based on the coordinates of the RT2K-Wo structure (protein and 2-phosphate drawn with tan bonds) and the coordinates of Fru-6-P from the H256A structure (Fru-6-P drawn with cyan bonds). We have not repositioned either the 2-phosphate or the Fru-6-P, so there is a small (0.9-Å) gap between the Fru-6-P-O-2 and the phosphate oxygen. This gap would obviously not exist in Fru-2,6-P[2], since these represent the same oxygen. The catalytic His256 is positioned in-line with the O-2-P bond, while Asn262, His390, and Arg255 are arranged perpindicular to that line, in a position to interact with the equatorial oxygens in the pentacoordinate transition state. Arg305 is not shown, to reduce the clutter in the figure but would be positioned in the foreground, interacting with the phosphate oxygen that is shown interacting with His390. Glu325 is shown hydrogen-bonded with Fru-6-P-O-2 and also interacting with the N terminus of helix 14 (labeled as 392 and 393). Ile^267 is included to clearly demonstrate the stacking interaction between this side chain and the Fru-6-P. The identity of the side chains are indicated with the one-letter amino acid designation and position in the RT2K protein sequence. This figure was produced using MOLSCRIPT (40) and rendered in Raster3D (41).
Figure 4.
Fig. 4. The "molecular ruler" of the Fru-2,6-Pase active site. Based on the position of Fru-6-P in the Fru-2,6-Pase active site, there is a 5.5-Å distance between the reactive nitrogen of the catalytic histidine and the 2-OH of Fru-6-P (shown as the dark bar at the top). This distance can accommodate either an E·Fru-2,6-P[2] or E-P·Fru-6-P complex but would preclude either an E-P·H[2]O·Fru-6-P or E·P·Fru-6-P complex. The dark bars below each complex represent ideal bond lengths or approximate Van der Waals contact distances.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (1999, 274, 2176-2184) copyright 1999.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19015259 H.Li, and G.Jogl (2009).
Structural and biochemical studies of TIGAR (TP53-induced glycolysis and apoptosis regulator).
  J Biol Chem, 284, 1748-1754.  
16008555 N.Chevalier, L.Bertrand, M.H.Rider, F.R.Opperdoes, D.J.Rigden, and P.A.Michels (2005).
6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis.
  FEBS J, 272, 3542-3560.  
11504626 A.Peracchi (2001).
Enzyme catalysis: removing chemically 'essential' residues by site-directed mutagenesis.
  Trends Biochem Sci, 26, 497-503.  
11514674 D.J.Rigden, I.Bagyan, E.Lamani, P.Setlow, and M.J.Jedrzejas (2001).
A cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus is actually a broad specificity phosphatase.
  Protein Sci, 10, 1835-1846.  
10933792 D.A.Okar, D.H.Live, M.H.Devany, and A.J.Lange (2000).
Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
  Biochemistry, 39, 9754-9762.  
11106417 J.Nairn, D.Duncan, N.E.Price, S.M.Kelly, L.A.Fothergill-Gilmore, S.Uhrinova, P.N.Barlow, D.J.Rigden, and N.C.Price (2000).
Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase. Elucidation of the roles of amino acids involved in substrate binding and catalysis.
  Eur J Biochem, 267, 7065-7074.  
11123954 M.Sakurai, P.F.Cook, C.A.Haseman, and K.Uyeda (2000).
Glutamate 325 is a general acid-base catalyst in the reaction catalyzed by fructose-2,6-bisphosphatase.
  Biochemistry, 39, 16238-16243.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time.