3QPV

PFKFB3 trapped in a phospho-enzyme intermediate state

3QPV の概要

• エントリーDOI: 10.2210/pdb3qpv/pdb
• 関連するPDBエントリー: 3QPU 3QPW
• 分子名称: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3, 2,6-di-O-phosphono-beta-D-fructofuranose, PHOSPHONIC ACID, ... (6 entities in total)
• 機能のキーワード: kinase/bisphosphatase, transferase, hydrolase
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 60803.59

構造登録者

Cavalier, M.C.,Kim, S.G.,Neau, D.,Lee, Y.H. (登録日: 2011-02-14, 公開日: 2012-02-08, 最終更新日: 2024-02-21)

主引用文献

Cavalier, M.C.,Kim, S.G.,Neau, D.,Lee, Y.H.
Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function.
Proteins, 80:1143-1153, 2012

PubMed Abstract: The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P(2)ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF(4)), and in a complex with pyrophosphate (PFKFB3•PP(i)) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3-P•F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF(4) and PFKFB3•PP(i) complexes were obtained by soaking. The PFKFB3•AlF(4) and PFKFB3•PP(i) complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ~5.1 Å, and the pivotal point 2-P is on the same line, suggesting an "in-line" transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3•PP(i), implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P(2) ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.

PubMed: 22275052
DOI: 10.1002/prot.24015

実験手法

X-RAY DIFFRACTION (2.5 Å)