3UQD

Crystal structure of the Phosphofructokinase-2 from Escherichia coli in complex with substrates and products

Summary for 3UQD

• Entry DOI: 10.2210/pdb3uqd/pdb
• Related: 3CQD 3N1C 3UQE
• Descriptor: 6-phosphofructokinase isozyme 2, ADENOSINE-5'-TRIPHOSPHATE, 6-O-phosphono-beta-D-fructofuranose, ... (7 entities in total)
• Functional Keywords: phosphofructokinases, pfk-2, glycolysis, transferase
• Biological source: Escherichia coli
• Total number of polymer chains: 4
• Total formula weight: 134177.06

Authors

Pereira, H.M.,Caniuguir, A.,Baez, M.,Cabrera, R.,Babul, J. (deposition date: 2011-11-20, release date: 2012-11-28, Last modification date: 2023-09-13)

Primary citation

Murillo-Lopez, J.,Zinovjev, K.,Pereira, H.,Caniuguir, A.,Garratt, R.,Babul, J.,Recabarren, R.,Alzate-Morales, J.,Caballero, J.,Tunon, I.,Cabrera, R.
Studying the phosphoryl transfer mechanism of theE. coliphosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations.
Chem Sci, 10:2882-2892, 2019

PubMed Abstract: Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from , which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.

PubMed: 30996866
DOI: 10.1039/c9sc00094a

Experimental method

X-RAY DIFFRACTION (2.14 Å)