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.

On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study

“…In order to investigate the catalytic mechanism, hybrid QM/MM procedure was applied as previously reported in similar systems [ 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. The QM region includes Cys18, His22 of the seven chains and Glu25 faced into the hydrophobic channel of the CC-Hept-Cys-His-Glu and three water molecules (w1, w2 and w3) engaged in a hydrogen bonds network (see Figure 4 ) both with each other and with the catalytic triad ( Figure S6 ).…”

“…In order to quantify entropic contributions, the Grimme’s procedure [ 50 ] was employed as reported in other works [ 14 , 15 , 16 ]. According to this method, only vibrational temperatures higher than 100 K are taken into account to calculate TΔS contribution using the harmonic oscillator/rigid rotor/particle in a box formalism.…”

“…According to this method, only vibrational temperatures higher than 100 K are taken into account to calculate TΔS contribution using the harmonic oscillator/rigid rotor/particle in a box formalism. The final free energy surfaces were obtained including electronic energies, calculated with the larger 6-311+G(2d,2p) basis set, the zero point energy (ZPE) corrections, entropy contributions and empirical-D3 dispersion corrections [ 51 ], as reported in other works [ 14 , 15 , 16 , 49 ]. The natural bond orbitals analysis (NBO) [ 52 ] as implemented in Gaussian 09 code was carried out on each stationary point intercepted along the free energy surfaces.…”

Exploring the Structure–Performance Relationship of Sulfonated Polysulfone Proton Exchange Membrane by a Combined Computational and Experimental Approach

“…According to experimental evidence, which indicates a membrane sulfonation level equal to 80%, the model contains 216 -SO 3 groups. A well assessed protocol, [23][24][25] based on the General Amber Force Field (GAFF) [26] and RESP method [27], was adopted to extrapolate Lennard-Jones and charges parameters, on previously HF/6-31G* optimized structures of each component of the polymer. The corresponding parameters are provided in AMBER format in the Supplementary Information.…”

“…Starting from the equilibrated structures obtained from a preliminary MD study on a single polymer model containing 72 monomers (see Figure S2 and Supporting Information for further details). The final geometry was obtained adopting the Packmol soft- A well assessed protocol, [23][24][25] based on the General Amber Force Field (GAFF) [26] and RESP method [27], was adopted to extrapolate Lennard-Jones and charges parameters, on previously HF/6-31G* optimized structures of each component of the polymer. The corresponding parameters are provided in AMBER format in the Supplementary Information.…”