How the Destabilization of a Reaction Intermediate Affects Enzymatic Efficiency: The Case of Human Transketolase

Atomic
resolution X-ray crystallography has shown that an intermediate
(the X5P-ThDP adduct) of the catalytic cycle of transketolase (TK)
displays a significant, putatively highly energetic, out-of-plane
distortion in a sp2 carbon
adjacent to a lytic bond, suggested to lower the barrier of the subsequent
step, and thus was postulated to embody a clear-cut demonstration
of the intermediate destabilization effect. The lytic
bond of the subsequent rate-limiting step was very elongated in the
X-ray structure (1.61 Å), which was proposed to be a consequence
of the out-of-plane distortion. Here we use high-level QM and QM/MM
calculations to study the intermediate destabilization effect. We show that the intrinsic energy penalty for the observed
distortion is small (0.2 kcal·mol–1) and that
the establishment of a favorable hydrogen bond within X5P-ThDP, instead
of enzyme steric strain, was found to be the main cause for the distortion.
As the net energetic effect of the distortion is small, the establishment
of the internal hydrogen bond (−0.6 kcal·mol–1) offsets the associated penalty. This makes the distorted structure
more stable than the nondistorted one. Even though the energy contributions
determined here are close to the accuracy of the computational methods
in estimating penalties for geometric distortions, our data show that
the intermediate destabilization effect provides
a small contribution to the observed reaction rate and does not represent
a catalytic effect that justifies the many orders of magnitude which
enzymes accelerate reaction rates. The results help to understand
the intrinsic enzymatic machinery behind enzyme’s amazing proficiency.