Energy Landscape of the Michaelis Complex of Lactate Dehydrogenase: Relationship to Catalytic Mechanism

Peng, Huo Lei; Deng, Hua; Dyer, R. Brian; Callender, Robert

doi:10.1021/bi500215a

Cited by 34 publications

(104 citation statements)

References 38 publications

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“…Thus, it suggests that the conformations in basin B are more reactive toward the hydride transfer step than those in basin A, the population of which is larger than that of basin B according to D–A distance analysis, which is consistent with the experimental results. 2 …”

Section: Resultsmentioning

confidence: 99%

Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Pan

Schwartz

2015

J. Phys. Chem. B

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It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

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Section: Resultsmentioning

confidence: 99%

Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Pan

Schwartz

2015

J. Phys. Chem. B

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“…25,29 Infrared cells consisted of CaF 2 windows with 15 μm Teflon spacers. Spectra were collected in the range 1100–4000 cm –1 with 2 cm –1 resolution.…”

Section: Methodsmentioning

confidence: 99%

Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy

et al. 2014

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Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm−1, respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions.

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“…Correlated with this work, Callender and co-workers have used isotope-edited difference Fourier transform infrared spectroscopy measurements on the NADH·pyruvate Michaelis complex for the wild type LDH and a mutant (Asp168Asn) with an impaired catalytic efficiency, suggesting that their results provide direct evidence of a restricted ensemble of more reactive conformational substates in the enzyme system than in the non-catalyzed reaction in solution. 11 Experiments of single-molecule enzymatic dynamics have been carried out on other systems such as flavin adenine dinucleotide (FAD), 12 or horseradish peroxidase (HRP). 13,14 The presence of a wide distribution of rates in individual enzyme molecules have also been predicted by computer simulations on different enzymatic systems, 15,16,17 including LDH.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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Energy Landscape of the Michaelis Complex of Lactate Dehydrogenase: Relationship to Catalytic Mechanism

Cited by 34 publications

References 38 publications

Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy

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

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