An enzyme in action seen by high-resolution quasielastic neutron scattering

Abir Nesrine Hassani a,b , Luman Haris b,c, Markus Appel d, Tilo Seydel d, Andreas M. Stadler b,c, Gerald R. Kneller a,e

a Centre de Biophysique Moléculaire, CNRS and Université d’Orléans, Rue Charles Sadron, 45071 Orléans, Franceb Jülich Centre for Neutron Science (JCNS-1) and Institute of Biological Information Processing (IBI-8), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany
c Institute of Physical Chemistry, RWTH Aachen University, Landoltweg 2, 52056 Aachen, Germany
d Institut Laue Langevin, 71 Avenue des Martyrs, 38042 Grenoble Cedex 9, France
e Laboratoire des biomolécules, Département de chimie, Ecole Normale Supérieure, 75005 Paris, France

Hassani et al., J. Chem. Phys. 159 (14), 141102 (2023)

Enzymes are biocatalysts which accelerate all biochemical reactions within the metabolism of living systems and the internal dynamics of these biological macromolecules is vital for their function. A prominent example is Phosphoglycerate Kinase (PGK), which is a monomeric two-domain protein catalyzing the transformation of adenosine diphosphate (ADP) into adenosine triphosphate (ATP) and thus the production of energy carriers within the living cell. Here the active site is a well-conserved hinge region which links the two domains of the protein (see Fig. 1). PGK has been studied with a variety of experimental and simulation techniques in order to elucidate the functional dynamics of this enzyme. We mention in particular studies with neutron spin echo (NSE) spectroscopy in combination with normal mode analysis and molecular dynamics (MD) simulation, which suggest that the catalytic function of the of PGK is enabled by large-amplitude hinge-bending motions [1,2]. Here we report on a recent study of PGK with high-resolution quasielastic neutron scattering (QENS) in which the data analysis with a “minimalistic” multi-scale model for protein dynamics in space and time shows directly that the enzymatic activity of the molecule is accompanied by inter-domain motions. The model describes the intra-domain dynamics in PGK by anomalous, non-markovian diffusion in configuration space and the dynamics on larger scales by normal Brownian diffusion. It is inspired by Frauenfelder’s picture of protein dynamics as a thermally activated hopping process on a complex energy landscape [3] and has been adapted to the description of neutron scattering experiments considering that the “energy landscape” is the ensemble of quantum mechanical energy levels of the sample [4]. In our model the inter-domain motions in PGK are taken into account by a scale-dependent diffusion coefficient, D(Q), where Q is an inverse length which is defined by the momentum transfer from the neutron to the sample. Fig. 2 shows D(Q) for PGK in a deuterated buffer solution in presence and absence of substrates (orange and blue dots, respectively), where the deuteration is used to mask the solvent. One clearly observes a modulation of D(Q) in presence of substrates. The maximum at Q = 1.2 Å -1 corresponds to a spatial resolution of , which is roughly the size of the hinge region in PGK, where the enzymatic reactions take place (see Fig. 1). In absence of the substrates, the effective diffusion constant is essentially constant and very close to the value of D = 5.1 × 10-3 Å2/ps which is obtained by inserting the hydrodynamic radius of RH=30.5 Å of PGK into the Stokes-Einstein relation for the diffusion coefficient of a sphere with that radius (grey sphere in Fig. 1). Our work demonstrates that high-resolution QENS is a very sensitive technique which is able to probe the functional dynamics of enzymes in action if appropriate models are used for the interpretation of the experimental data.

Fig. 1: The studied molecule, Phosphoglycerate Kinase (PGK), with a sphere of radius 30.5 Å.
Fig. 2 : Diffusion coefficient as a function of Q determined from Quasielastic Neutron Scattering.

[1] R. Inoue et al. Biophysical Journal 99 (7), 2309–2317 (2010).
[2] N. Smolin et al., Biophysical Journal 102 (5), 1108–1117 (2012).
[3] H. Frauenfelder, S. G. Sligar, and P. G. Wolynes, Science 254 (5038), 1598– 1603 (1991).
[4] G. R. Kneller, PNAS USA 115 (38), 9450–9455 (2018).

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