Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase

Abstract

In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic structure, the free energy barrier for the transformation of pyruvate into lactate with the open conformation of the protein varies between 12.9 and 16.3 kcal/mol, after quantizing the vibrations and adding the contributions of recrossing and tunneling effects. These values are very close to the experimentally deduced one (14.2 kcal·mol−1) and ~2 kcal·mol−1 smaller than the ones obtained with the closed loop conformer. Calculation of primary KIEs and IR spectra in both protein conformations are also consistent with our hypothesis and in agreement with experimental data. Our calculations suggest that the closure of the active site is mainly required for the inverse process; the oxidation of lactate to pyruvate. According to this hypothesis H4 type LDH enzyme molecules, where it has been propose that lactate is transformed into pyruvate, should have a better ability to close the mobile loop than the M4 type LDH molecules.

Alternative Mechanistic Strategy for Enzyme Catalysis in a Ni‐Dependent Lactate Racemase (LarA): Intermediate Destabilization by the Cofactor

“…The TPSSh‐D3 method yields a lower energy barrier of 14.6 kcal mol −1 , while the widely‐used B3LYP‐D3 method predicts a barrier of 17.8 kcal mol −1 . Comparable or slightly lower barriers (15.0–19.0 kcal mol −1 ) have been reported in a recent QM/MM study on LDH with inclusion of the whole LDH protein and a tunneling effect . During the dehydrogenation process, the negative charge of the hydride and the positive charge of the proton principally transfer to the pyridinium ring and to the nearby histidine base, respectively (Table ).…”

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

“…Despite there are precedents on the formation of stable ion pair stationary state structures before peptide bond breaking in high-level DFT PESs, 115 the change in the topology of PESs or free energy surfaces after high level QM/MM corrections is an effect that has been already observed in previous studies. 116, 117, 118 In particular, intermediates located for enzyme catalyzed reactions involving a double proton and hydride transfers, when the QM subset of atoms were described by a semiempirical method, were transformed into just a shallow minimum when improving the description of the QM region at DFT level, 116 or even disappeared when the correction was at DFT 117 or MP2 118 level. Then, it is reasonable to believe that high level M06-2X/MM QM/MM MD trajectories could perfectly predict enzyme KIE that would fit within the two values obtained at AM1/MM level, supporting a reaction mechanism through a GD intermediate as shown in Figure 6.…”

The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism

“…The presence of a wide distribution of rates in individual enzyme molecules have also been predicted by computer simulations on different enzymatic systems. 10,43,44,45,46,47 …”