posted on 2023-11-24, 14:33authored bySneha Sahu, Sayani Ghosh, Sushant K. Sinha, Supratim Datta, Neelanjana Sengupta
A crucial
prerequisite for industrial applications of
enzymes is
the maintenance of specific activity across wide thermal ranges. β-Glucosidase
(EC 3.2.1.21) is an essential enzyme for converting cellulose in biomass
to glucose. While the reaction mechanisms of β-glucosidases
from various thermal ranges (hyperthermophilic, thermophilic, and
mesophilic) are similar, the factors underlying their thermal sensitivity
remain obscure. The work presented here aims to unravel the molecular
mechanisms underlying the thermal sensitivity of the enzymatic activity
of the β-glucosidase BglB from the bacterium Paenibacillus
polymyxa. Experiments reveal a maximum enzymatic activity
at 315 K, with a marked decrease in the activity below and above this
temperature. Employing in silico simulations, we
identified the crucial role of the active site tunnel residues in
the thermal sensitivity. Specific tunnel residues were identified
via energetic decomposition and protein–substrate hydrogen
bond analyses. The experimentally observed trends in specific activity
with temperature coincide with variations in overall binding free
energy changes, showcasing a predominantly electrostatic effect that
is consistent with enhanced catalytic pocket–substrate hydrogen
bonding (HB) at Topt. The entropic advantage
owing to the HB substate reorganization was found to facilitate better
substrate binding at 315 K. This study elicits molecular-level insights
into the associative mechanisms between thermally enabled fluctuations
and enzymatic activity. Crucial differences emerge between molecular
mechanisms involving the actual substrate (cellobiose) and a commonly
employed chemical analogue. We posit that leveraging the role of fluctuations
may reveal unexpected insights into enzyme behavior and offer novel
paradigms for enzyme engineering.