Issue 10, 2019

Studying the phosphoryl transfer mechanism of the E. coli phosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations

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 E. coli, 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 Mg2+. 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.

Graphical abstract: Studying the phosphoryl transfer mechanism of the E. coli phosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations

Supplementary files

Article information

Article type
Edge Article
Submitted
07 yan 2019
Accepted
24 yan 2019
First published
28 yan 2019
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2019,10, 2882-2892

Studying the phosphoryl transfer mechanism of the E. coli phosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations

J. Murillo-López, K. Zinovjev, H. Pereira, A. Caniuguir, R. Garratt, J. Babul, R. Recabarren, J. Alzate-Morales, J. Caballero, I. Tuñón and R. Cabrera, Chem. Sci., 2019, 10, 2882 DOI: 10.1039/C9SC00094A

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