1 research outputs found
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