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Homodimerization of a glycoside hydrolase family GH1 β‐glucosidase suggests distinct activity of enzyme different states
Author(s) -
Otsuka Felipe A. M.,
Chagas Rafael S.,
Almeida Vitor M.,
Marana Sandro R.
Publication year - 2020
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3908
Subject(s) - dimer , chemistry , monomer , dissociation constant , conformational change , size exclusion chromatography , dynamic light scattering , dissociation (chemistry) , reaction rate constant , enzyme , stereochemistry , equilibrium constant , kinetics , chromatography , biophysics , biochemistry , organic chemistry , polymer , materials science , receptor , physics , quantum mechanics , nanoparticle , nanotechnology , biology
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.