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 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.
Highlights
Enzymes play an essential role in a broad variety of biochemical processes
A resulting aspect is that a substrate that binds the enzyme in a conformation that looks like the transition state needs to climb a lower barrier to reach the transition state, increasing kcat.[16−18] This very interesting proposal is not free from controversy, as the kcat increase might be achieved at the cost of increasing KM as well, and it is not clear how the ground-state destabilization does increase the enzyme efficiency, which is the relevant rate constant in physiologic conditions.[13]
The molecular model used for the quantum mechanical/molecular mechanical (QM/MM) calculations was obtained starting from the X-ray structure of Human transketolase (hTK) complexed with the thiamine diphosphate (ThDP)-X5P adduct, at the resolution of 0.97 Å (PDB id: 4KXV), isolated from Homo sapiens.[20]
Summary
Enzymes play an essential role in a broad variety of biochemical processes. Understanding these processes is an interest, and a major challenge, for the research community.[1,2] The most popular theory about the origin of enzyme’s catalytic power was proposed by Pauling in 1948.3 The underlying idea is that enzymes catalyze reactions by binding better the transition state than the ground state, which is materialized through a higher binding affinity for the former. The proposal suggests that the enzyme rate constant (kcat), and the enzyme’s efficiency (kcat/KM), is very dependent on the substrate conformation in the Michaelis complex.[14,15] A resulting aspect is that a substrate that binds the enzyme in a conformation that looks like the transition state (which can be seen as “distorted” when compared to the lower-energy aqueous solution conformation) needs to climb a lower barrier to reach the transition state, increasing kcat.[16−18] This very interesting proposal is not free from controversy, as the kcat increase might be achieved at the cost of increasing KM as well, and it is not clear how the ground-state destabilization does increase the enzyme efficiency (kcat/KM), which is the relevant rate constant in physiologic conditions.[13] The case of the intermediate destabilization is different, as the substrate still binds the enzyme in the relaxed conformation (without increasing KM) but is “protected” from falling into low-energy intermediates (through enzyme-induced distortion) that would trap it in the bottom of high-barrier wells In this sense, the intermediate destabilization might increase kcat without increasing KM
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