PDBsum entry: 2bif

Transferase, hydrolase

PDB id: 2bif

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

Protein chains

P25114  (F264_RAT) -  6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4

Seq: 469 a.a.

Struc: 432 a.a.*

* 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.
• 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.
• 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 

• Biological process: fructose metabolic process (2 terms)
• Biochemical function: catalytic activity (9 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 the author.