Principles of Cold Adaptation of Fish Lactate Dehydrogenases Revealed by Computer Simulations of the Catalytic Reaction

Abstract

Cold-adapted enzymes from psychrophilic and psychrotolerant species are characterized by a higher catalytic activity at low temperature than their mesophilic orthologs and are also usually found to be more thermolabile. Computer simulations of the catalytic reactions have been shown to be a very powerful tool for analyzing the structural and energetic origins of these effects. Here, we examine the cold-adaptation of lactate dehydrogenases from two Antarctic and sub-Antarctic fish species using this approach and compare our results to those obtained for the orthologous dogfish enzyme. Direct calculations of thermodynamic activation parameters show that the cold-adapted fish enzymes are characterized by a lower activation enthalpy and a more negative entropy term. This appears to be a universal feature of psychrophilic enzymes and it is found to originate from a higher flexibility of certain parts of the protein surface. We also carry out free energy simulations that address the differences in thermal stability and substrate binding affinity between the two cold-adapted enzymes, which only differ by a single mutation. These calculations capture the effects previously seen in in vitro studies and provide straightforward explanations of these experimental results.

Reply to Comment on: “Computer Simulations Reveal an Entirely Entropic Activation Barrier for the Chemical Step in a Designer Enzyme”

“…Hence, all of the loops covering the active site are fully mobile and it is, in fact, only 163 atoms (belonging to 16 residues) on the back side of the enzyme that are restrained. This type of simulation model has been shown earlier for numerous systems to give accurate values of thermodynamic activation parameters compared to experiment. − In particular, the model has also been shown to capture the curved Arrhenius plot for a cold-adapted α-amylase, demonstrating that the curvature is not due to a heat capacity effect but to the existence of an inactive state of the ES complex . This has recently also been verified by us experimentally by redesign of the α-amylase .…”

Acclimation to warm temperatures modulates lactate and malate dehydrogenase isozymes in juvenile Horabagrus brachysoma (Günther)

“…LDH is the most widely researched enzyme in fish. It is a cytosolic enzyme that catalyses the interconversion of lactate to pyruvate in the final step of anaerobic glycolysis and also the conversion of lactate to glucose during gluconeogenesis (Zakhartsev et al 2004;Koenekoop and Åqvist 2023). It is a tetrameric protein that is controlled by three distinct genes viz., LDH-A*, LDH-B*, and LDH-C*.…”

Why Do Empirical Valence Bond Simulations Yield Accurate Arrhenius Plots?

“…Among the examples given above, we have representatives of all four variants of EVB parametrization (solution experiments, 10 solution DFT, 3,4,7 enzyme cluster DFT, 9 and enzyme QM/MM DFT) 5,6 and the predicted values of ΔH ⧧ and ΔS ⧧ are in all cases remarkably good.…”

“…For a number of examined enzyme reactions, we found that the calculated thermodynamic activation parameters are in remarkably good agreement with experimental measurements. Some recent examples of this are cytidine deaminase, 9 EF-Tu catalyzed GTP hydrolysis on the ribosome, 10 α-amylase, 4 hydroxybutyrate dehydrogenase, 5 lactate dehydrogenase, 6 and chorismate mutase. 7 The most recent case of chorismate mutase is particularly instructive, since it is a unimolecular reaction in the enzyme (and in solution), with no enzymic groups participating in the actual chemistry.…”

“…The empirical valence bond (EVB) method , has proven to be very useful for calculating thermodynamic activation parameters for chemical reactions, both in solution and in enzymes. − In 2008, we devised a procedure analogous to experimental Arrhenius plots to obtain activation free energies as a function of temperature from molecular dynamics (MD) simulations in combination with the EVB method . With high enough precision for the calculated activation free energies, computational Arrhenius plots of either Δ G ⧧ / T vs 1/ T or Δ G ⧧ vs T can thus be constructed, which allows the values of Δ H ⧧ and Δ S ⧧ to be reliably extracted by linear regression.…”