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

Atomic resolution X-ray crystallography has shownthat an intermediate (the X5P-ThDP adduct) of the catalytic cycleof transketolase (TK) displays a significant, putatively highlyenergetic, out-of-plane distortion in a sp 2 carbon adjacent to a lyticbond, suggested to lower the barrier of the subsequent step, andthus was postulated to embody a clear-cut demonstration of theintermediate destabilization effect. The lytic bond of the subsequentrate-limiting step was very elongated in the X-ray structure (1.61Å), which was proposed to be a consequence of the out-of-planedistortion. Here we use high-level QM and QM/MM calculationsto study the intermediate destabilization effect. We show that theintrinsic energy penalty for the observed distortion is small (0.2kcal·mol−1) and that the establishment of a favorable hydrogen bond within X5P-ThDP, instead of enzyme steric strain, was found tobe the main cause for the distortion. As the net energetic effect of the distortion is small, the establishment of the internal hydrogenbond (−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 estimatingpenalties for geometric distortions, our data show that the intermediate destabilization effect provides a small contribution to theobserved reaction rate and does not represent a catalytic effect that justifies the many orders of magnitude which enzymes acceleratereaction rates. The results help to understand the intrinsic enzymatic machinery behind enzyme’s amazing proficiency.