PDBsum entry 2i1v

Transferase, hydrolase

PDB id: 2i1v

Contents

Protein chain

449 a.a. *

Ligands

FDP

F6P

ADP

PHS

Waters ×222

* Residue conservation analysis

PDB id:

2i1v

Name:

Transferase, hydrolase

Title:

Crystal structure of pfkfb3 in complex with adp and fructose-2,6- bisphosphate

Structure:

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3. Chain: b. Synonym: pfkfb3, phosphoryl transferase. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.50Å R-factor: 0.214 R-free: 0.262

Authors:

S.G.Kim,M.R.El-Maghrabi,Y.H.Lee

Key ref:

S.G.Kim et al. (2007). A Direct Substrate-Substrate Interaction Found in the Kinase Domain of the Bifunctional Enzyme, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase. J Mol Biol, 370, 14-26. PubMed id: 17499765 DOI: 10.1016/j.jmb.2007.03.038

Date:

15-Aug-06 Release date: 03-Jul-07

Protein chain

Q16875  (F263_HUMAN) -  6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 from Homo sapiens

Seq: 520 a.a.

Struc: 449 a.a.

Enzyme reactions

• Enzyme class 2: E.C.2.7.1.105 - 6-phosphofructo-2-kinase.
• Reaction: beta-D-fructose 6-phosphate + ATP = beta-D-fructose 2,6-bisphosphate + ADP + H⁺

beta-D-fructose 6-phosphate + ATP = beta-D-fructose 2,6-bisphosphate (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP (Bound ligand (Het Group name = FDP) corresponds exactly) + H(+)

• Enzyme class 3: E.C.3.1.3.46 - fructose-2,6-bisphosphate 2-phosphatase.
• Reaction: beta-D-fructose 2,6-bisphosphate + H2O = beta-D-fructose 6-phosphate + phosphate

beta-D-fructose 2,6-bisphosphate (Bound ligand (Het Group name = FDP) corresponds exactly) + H2O = beta-D-fructose 6-phosphate + phosphate (Bound ligand (Het Group name = PHS) matches with 80.00% similarity)

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

reference

DOI no: 10.1016/j.jmb.2007.03.038

J Mol Biol 370:14-26 (2007)

PubMed id: 17499765

A Direct Substrate-Substrate Interaction Found in the Kinase Domain of the Bifunctional Enzyme, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase.

S.G.Kim, M.Cavalier, M.R.El-Maghrabi, Y.H.Lee.

ABSTRACT

To understand the molecular basis of a phosphoryl transfer reaction catalyzed by the 6-phosphofructo-2-kinase domain of the hypoxia-inducible bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), the crystal structures of PFKFB3AMPPCPfructose-6-phosphate and PFKFB3ADPphosphoenolpyruvate complexes were determined to 2.7 A and 2.25 A resolution, respectively. Kinetic studies on the wild-type and site-directed mutant proteins were carried out to confirm the structural observations. The experimentally varied liganding states in the active pocket cause no significant conformational changes. In the pseudo-substrate complex, a strong direct interaction between AMPPCP and fructose-6-phosphate (Fru-6-P) is found. By virtue of this direct substrate-substrate interaction, Fru-6-P is aligned with AMPPCP in an orientation and proximity most suitable for a direct transfer of the gamma-phosphate moiety to 2-OH of Fru-6-P. The three key atoms involved in the phosphoryl transfer, the beta,gamma-phosphate bridge oxygen atom, the gamma-phosphorus atom, and the 2-OH group are positioned in a single line, suggesting a direct phosphoryl transfer without formation of a phosphoenzyme intermediate. In addition, the distance between 2-OH and gamma-phosphorus allows the gamma-phosphate oxygen atoms to serve as a general base catalyst to induce an "associative" phosphoryl transfer mechanism. The site-directed mutant study and inhibition kinetics suggest that this reaction will be catalyzed most efficiently by the protein when the substrates bind to the active pocket in an ordered manner in which ATP binds first.

Selected figure(s)

Figure 3.
Figure 3. A cartoon of the suggested catalytic pathway. In the clockwise direction, 2-OH of Fru-6-P is deprotonated by the γ-phosphate moiety of ATP and a nucleophilic attack occurs. An intermediate pentavalent phosphorane is formed, and the negative charges generated on the planary oxygen atoms are stabilized by Lys47, Lys168, and Mg^2+, and maybe a H^+ extracted from 2-OH. Redistribution of the phosphorane electrons breaks a diester bond between the bridge oxygen and γ-phosphorus atoms. The leaving group is stabilized by a H^+. Eventually, the leaving group (ADP) is stabilized through a salt-bridge to Lys168 to finish the reaction.

Figure 4.
Figure 4. PEP binding to PFKFB3. (a) The |F[o]|–|F[c]| omit electron density map. The map is calculated in the absence of ligands and contoured at 2.5σ. (b) A stereo view of the interactions of PEP with the 2-Kase active site pocket is shown. The dotted lines represent hydrogen bonds or salt-bridges. To show its position in the 2-Kase active pocket, the structure of PFKFB3 radical dot AMPPCP radical dot Fru-6-P complex is superposed and Fru-6-P (light gray) is shown.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 370, 14-26) copyright 2007.

Figures were selected by an automated process.