Thermodynamic and Structural Adaptation Differences between the Mesophilic and Psychrophilic Lactate Dehydrogenases

Abstract

The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mesophile), and mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay that can distinguish protein conformational changes close to and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) was surprisingly found to coincide with a marked change in enzymatic activity for both LDHs. A similar reversible structural transition of the active site structure was observed for phLDH but not for cgLDH. An observed lower substrate binding affinity for cgLDH compared to that for phLDH was accompanied by a larger contribution of entropy to ΔG, which reflects a higher functional plasticity of the psychrophilic cgLDH compared to that of the mesophilic phLDH. The natural osmolyte, trimethylamine N-oxide (TMAO), increases stability and shifts all structural transitions to higher temperatures for both orthologs while simultaneously reducing catalytic activity. The presence of TMAO causes cgLDH to adopt catalytic parameters like those of phLDH in the absence of the osmolyte. Our results are most naturally understood within a model of enzyme dynamics whereby different conformations of the enzyme that have varied catalytic parameters (i.e., binding and catalytic proclivity) and whose population profiles are temperature-dependent and influenced by osmolytes interconvert among themselves. Our results also show that adaptation can be achieved by means other than gene mutations and complements the synchronic evolution of the cellular milieu.

Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Förster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase

“…10 Furthermore, pig heart LDH demonstrates a structural change and similar break in enzyme activity around 35°C, indicating that elevated glass transitions may not be restricted to thermophilic enzymes. 18…”

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178

“…rPsLeuDH also exhibited lower Δ H , Δ S and Δ G and higher k cat at low temperature, as compared to mesophilic enzyme, which may be mainly related to the conformation of cold adapted protein [ 27 ]. On the other hand, it may also be related to increasing the efficiency of binding of the substrate to the catalytic site [ 28 ].…”

Pressure tolerance of deep‐sea enzymes can be evolved through increasing volume changes in protein transitions: a study with lactate dehydrogenases from abyssal and hadal fishes

“…Rapid kinetics experiments and molecular dynamics simulations demonstrated that the LDH complex with NADH exists in quite a range of protein conformations with different degree of openness and solvation of the active site, whereby only a minority population of the enzyme is binding competent [50,51]. Once the enzyme-substrate complex is formed, its further catalytic conversion also proceeds through several different trajectories-the active site conformation of LDH is shown to exhibit important heterogeneity, so that the LDH reaction landscape branches at multiple points creating pathways with varied reactivity [51,73,75,76]. Importantly, the conformational transitions that control the rate of substrate binding and determine the partitioning between different catalytic branches are all associated with substantial changes in protein solvation and, consequently, large volume changes.…”

“…Importantly, the conformational transitions that control the rate of substrate binding and determine the partitioning between different catalytic branches are all associated with substantial changes in protein solvation and, consequently, large volume changes. It makes the catalytic landscape of LDH particularly susceptible to hydrostatic pressure [77] and addition of osmolytes, such as TMAO [52,75].…”

“…Increase in osmotic pressure via addition of cosolvents is known to have a profound effect on the activity and stability of LDH. Thus, addition of TMAO was shown to increase the thermal stability of LDH from the mackerel icefish (Champsocephalus gunnari L€ onnberg 1905) muscle, while simultaneously reducing its catalytic activity [75]. A study with LDH from rabbit muscle showed a displacement of P ½ of its inactivation to higher pressure by an addition of TMAO.…”