posted on 2015-07-27, 00:00authored byXiangfei Song, Yefei Wang, Shujun Zhang, Shihai Yan, Tong Li, Lishan Yao
An
attempt is made to evaluate the dielectric constant of the Trichoderma reesei Cel7B active site. Through kinetic measurements,
the pKa value of the catalytic acid E201
is determined. Mutations (away from E201) with net charge changes
are introduced to perturb the E201 pKa. It is shown that the mutation with a +1 charge change (including
G225R, G230R, and A335R) decreases the pKa of E201, whereas the mutation with a −1 charge change (including
Q149E, A222D, G225D, and G230D) increases the pKa. This effect is consistent with the electrostatic interaction
between the changed charge and the E201 side chain. The fitting of
the experimental data yields an apparent dielectric constant of 25–80.
Molecular dynamics simulations with explicit water molecules indicate
that the high solvent accessibility of the active site contributes
largely to the high dielectric constant. ONIOM calculations show that
high dielectric constant benefits the catalysis through decreasing
the energy of the transition state relative to that of the enzyme
substrate complex.