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

The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (<i>Sus scrofa</i>; a mesophile), and mackerel icefish, cgLDH (<i>Chamapsocephalus gunnari</i>; 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 Δ<i>G</i>, which reflects a higher functional plasticity of the psychrophilic cgLDH compared to that of the mesophilic phLDH. The natural osmolyte, trimethylamine <i>N</i>-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

Mechanism of Thermal Adaptation in the Lactate Dehydrogenases

The mechanism of thermal adaptation of enzyme function at the molecular level is poorly understood but is thought to lie within the structure of the protein or its dynamics. Our previous work on pig heart lactate dehydrogenase (phLDH) has determined very high resolution structures of the active site, via isotope edited IR studies, and has characterized its dynamical nature, via laser-induced temperature jump (T-jump) relaxation spectroscopy on the Michaelis complex. These particular probes are quite powerful at getting at the interplay between structure and dynamics in adaptation. Hence, we extend these studies to the psychrophilic protein cgLDH (<i>Champsocephalus gunnari</i>; 0 °C) and the extreme thermophile tmLDH (<i>Thermotoga maritima</i> LDH; 80 °C) for comparison to the mesophile phLDH (38−39 °C). Instead of the native substrate pyruvate, we utilize oxamate as a nonreactive substrate mimic for experimental reasons. Using isotope edited IR spectroscopy, we find small differences in the substate composition that arise from the detailed bonding patterns of oxamate within the active site of the three proteins; however, we find these differences insufficient to explain the mechanism of thermal adaptation. On the other hand, T-jump studies of reduced β-nicotinamide adenine dinucleotide (NADH) emission reveal that the most important parameter affecting thermal adaptation appears to be enzyme control of the specific kinetics and dynamics of protein motions that lie along the catalytic pathway. The relaxation rate of the motions scale as cgLDH > phLDH > tmLDH in a way that faithfully matches <i>k</i>_cat of the three isozymes

An example of parallel named entities in HTML in Chinese (a) and English (b).

<p>An example of parallel named entities in HTML in Chinese (a) and English (b).</p

Highest-ranked contextual patterns for the three classes (XXX means entity).

<p>Highest-ranked contextual patterns for the three classes (XXX means entity).</p

The diagram block of Chinese seed thesauruses extraction.

<p>The diagram block of Chinese seed thesauruses extraction.</p

Information after expanding thesauruses.

<p>Weight alg means the combined algorithm.</p

The HTML text of the list in Figure 3 in Chinese (a) and English (b).

<p>The HTML text of the list in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067526#pone-0067526-g003" target="_blank">Figure 3</a> in Chinese (a) and English (b).</p

Performance from BaiduBaike, baseline and Weight algorithm in the I2B2 corpus (%).

<p>Weight alg means the combined algorithm.</p

The upper graph depicts the baseline algorithm while the lower graph depicts the improved algorithm.

<p>The upper graph depicts the baseline algorithm while the lower graph depicts the improved algorithm.</p

Some of the extracted candidate lists from the webpage in Figure 3 in Chinese (a) and English (b).

<p>Some of the extracted candidate lists from the webpage in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067526#pone-0067526-g003" target="_blank">Figure 3</a> in Chinese (a) and English (b).</p