Large Scale Dynamics of the Michaelis Complex in <i>Bacillus stearothermophilus</i> Lactate Dehydrogenase Revealed by a Single-Tryptophan Mutant Study

Nie, Beining; Deng, Hua; Desamero, Ruel Z. B.; Callender, Robert

doi:10.1021/bi3017125

Cited by 14 publications

(41 citation statements)

References 29 publications

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“…To help elucidate the origins of these fast and slow transients, we measured the tryptophan fluorescence T -jump traces for the G106W mutant bsLDH· NADH complex (data not shown), which directly measure structural changes associated with the mobile loop. 18 Not surprisingly, the trace yielded a fit to a double exponential with a set of fast and slow transients that have rate constants of ~2000 and ~10000–20000 s −1 , respectively. This is significant because the structural changes as reported by the modulation of the NADH signal reflect a change in the loop tryptophan fluorescence.…”

Section: Resultsmentioning

confidence: 94%

“…Free concentrations were calculated using NADH dissociation constants obtained from steady-state fluorescence data. 18 Two distinct patterns emerge at low (<20 μM) and high (>100 μM) concentrations. At low concentrations, there is a sharp rise in the observed tryptophan relaxation rate and midrange NADH relaxation rate, and at higher concentrations, a much slower, nearly linear rise is observed.…”

Section: Resultsmentioning

confidence: 98%

“…14 Some studies were performed on a mutant bsLDH containing a single Trp residue, G106W, in which the three naturally occurring Trp residues have been mutated to tyrosines. 18 …”

Section: Methodsmentioning

confidence: 99%

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Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase fromBacillus stearothermophilus

et al. 2016

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Laser-induced temperature-jump relaxation spectroscopy was used to study the active site mobile-loop dynamics found in the binding of the NADH nucleotide cofactor and oxamate substrate mimic to lactate dehydrogenase in Bacillus stearothermophilus thermophilic bacteria (bsLDH). The kinetic data can be best described by a model in which NADH can bind only to the open-loop apoenzyme, oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open–closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed. The observed active-loop dynamics for bsLDH is very similar to that previously observed for pig heart LDH.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 98%

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Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase fromBacillus stearothermophilus

et al. 2016

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“…NADH fluorescence passed through a 458 nm narrow-band filter with the bandwidth of 40 nm full width at half-maximum (fwhm) before reaching the detector (Andover, Salem, NH). 19 …”

Section: Methodsmentioning

confidence: 99%

“…Creation of the pocket is accompanied by the motions of mobile areas within the protein, rearranging the pocket geometry to allow for favorable interactions between the cofactor and the ligand that facilitate on-enzyme catalysis. 19 Of particular interest to this work are the hydrogen bonds formed between Arg-109 and His-195 to the C2 carbonyl of pyruvate (emphasized in red). These bonds dictate the polarity of the carbonyl when pyruvate is bound.…”

Section: Figurementioning

confidence: 99%

Mechanism of Thermal Adaptation in the Lactate Dehydrogenases

et al. 2015

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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 (Champsocephalus gunnari; 0 °C) and the extreme thermophile tmLDH (Thermotoga maritima 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 kcat of the three isozymes.

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Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase

Reddish

Callender

Dyer

2017

Biophysical Journal

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Enzymes are known to exhibit conformational flexibility. An important consequence of this flexibility is that the same enzyme reaction can occur via multiple reaction pathways on a reaction landscape. A model enzyme for the study of reaction landscapes is lactate dehydrogenase. We have previously used temperature-jump (T-jump) methods to demonstrate that the reaction landscape of lactate dehydrogenase branches at multiple points creating pathways with varied reactivity. A limitation of this previous work is that the T-jump method makes only small perturbations to equilibrium and may not report conclusively on all steps in a reaction. Therefore, interpreting T-jump results of lactate dehydrogenase kinetics has required extensive computational modeling work. Rapid mixing methods offer a complementary approach that can access large perturbations from equilibrium; however, traditional enzyme mixing methods like stopped-flow do not allow for the observation of fast protein dynamics. In this report, we apply a microfluidic rapid mixing device with a mixing time of <100 μs that allows us to study these fast dynamics and the catalytic redox step of the enzyme reaction. Additionally, we report UV absorbance and emission T-jump results with improved signal-to-noise ratio at fast times. The combination of mixing and T-jump results yields an unprecedented view of lactate dehydrogenase enzymology, confirming the timescale of substrate-induced conformational change and presence of multiple reaction pathways.

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Large Scale Dynamics of the Michaelis Complex in Bacillus stearothermophilus Lactate Dehydrogenase Revealed by a Single-Tryptophan Mutant Study

Cited by 14 publications

References 29 publications

Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase fromBacillus stearothermophilus

Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase fromBacillus stearothermophilus

Mechanism of Thermal Adaptation in the Lactate Dehydrogenases

Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase

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