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

Abstract

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 sp ² 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.