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><sub>cat</sub> of the three isozymes
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