Leytens, Alexandre
[UCL]
Soumillion, Patrice
[UCL]
Alsteens, David
[UCL]
Over the last decades, significant advances have been achieved in the study of molecular motions of enzymes. The development of novel techniques allowed a deeper understanding of the dynamic processes underlying enzymatic catalysis at the molecular scale. It is now clear that proteins not only move, but some of their motions are crucial to their function. Moreover, recent studies would suggest that catalytically active enzyme diffuse faster in solution than when they rest. These studies, however, are not unanimously agreed upon and it is clear that some aspects of enzyme catalysis remain unknown. In parallel, atomic force microscopy (AFM) saw an important development in its applications especially in biological research. Single-molecule force spectroscopy (SMFS) techniques have been increasingly used for the study of proteins and other biomolecules. However, this technique has never been applied on catalytically active enzyme. With a vertical resolution in the Ångström scale and the ability to measure forces in real time in the pico Newton range, AFM appears to be an appropriated technology for the study of motions and forces involved in enzyme catalysis. To perform these experiments, Escherichia coli’s isocitrate dehydrogenase (EcIDH) was used as a model enzyme. EcIDH is a homodimeric enzyme catalysing a reaction of the Krebs cycle. It has the particularity of having both its N-termini pointing away from each other, outside of the tertiary structure of the enzyme and far from its catalytic sites. The N-termini of these enzymes was tagged in order to hold it under a constant force in AFM (Force clamp). This single-molecule approach could be used to investigate the behaviour of catalytically active or resting enzyme. In the presence of its substrates, the enzyme periodically produced oscillations of several nanometres during a few seconds. The frequency at which these oscillations could be observed varied upon substrate concentration, and the frequency of the oscillations as well as the energy it produced against the AFM tip slightly changed in the presence of the substrate, when compared to catalytically inactive controls. In addition to these force-clamp experiments, enzyme variants were produced and characterized in order to obtain a wider range of catalytic activity to serve as references. The residue Ser113 was chosen as a target for mutagenesis, and the enzymes were characterized using UV-spectrophotometry. The construction of these mutants also allowed the verification that subunits exchange occurs between different EcIDH variants; and thus, leads to the formation of heterodimers. To help elucidate the origin of the observed oscillations, chemical crosslinking of EcIDH subunits was performed. For the first time, force clamp spectroscopy was performed on a catalytically active enzyme. The obtained results suggest a link between the observed oscillations and the catalytic activity of the enzyme. While the exact mechanism behind these oscillations could not yet be determined, these primary results demonstrate the potential of the application of this technique to the study of enzyme catalysis.
Bibliographic reference |
Leytens, Alexandre. Probing the catalytic activity of E. coli isocitrate dehydrogenase by force-clamp spectroscopy. Faculté des sciences, Université catholique de Louvain, 2020. Prom. : Soumillion, Patrice ; Alsteens, David. |
Permanent URL |
http://hdl.handle.net/2078.1/thesis:23042 |