Homodimerization of a glycoside hydrolase family <scp>GH1</scp> β‐glucosidase suggests distinct activity of enzyme different states

Abstract

In this work, we investigated how activity and oligomeric state are related in a purified GH1 β-glucosidase from Spodoptera frugiperda (Sfβgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (K_D ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfβgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (k_off ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (k_obs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfβgly concentration. These data indicated that Sfβgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 β-glucosidases, but it can also help to elucidate protein interaction pathways.