2 research outputs found
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><sub>cat</sub> of the three isozymes
Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy
Protein
conformational heterogeneity and dynamics are known to
play an important role in enzyme catalysis, but their influence has
been difficult to observe directly. We have studied the effects of
heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase
using isotope edited infrared spectroscopy, laser-induced temperature
jump relaxation, and kinetic modeling. The isotope edited infrared
spectrum reveals the presence of multiple reactive conformations of
pyruvate bound to the enzyme, with three major reactive populations
having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm<sup>–1</sup>, respectively. The temperature jump relaxation measurements
and kinetic modeling indicate that these substates form a heterogeneous
branched reaction pathway, and each substate catalyzes the conversion
of pyruvate to lactate with a different rate. Furthermore, the rate
of hydride transfer is inversely correlated with the frequency of
the C2 carbonyl stretch (the rate increases as the frequency decreases),
consistent with the relationship between the frequency of this mode
and the polarization of the bond, which determines its reactivity
toward hydride transfer. The enzyme does not appear to be optimized
to use the fastest pathway preferentially but rather accesses multiple
pathways in a search process that often selects slower ones. These
results provide further support for a dynamic view of enzyme catalysis
where the role of the enzyme is not just to bring reactants together
but also to guide the conformational search for chemically competent
interactions