Conformational Heterogeneity within the Michaelis Complex of Lactate Dehydrogenase

Deng, Hua; Vu, Dung M.; Clinch, Keith; Desamero, Ruel Z. B.; Dyer, R. Brian; Callender, Robert

doi:10.1021/jp2015929

Cited by 30 publications

(68 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this kinetic scheme, which is in agreement with various results, 7,8,15 the encounter complex, presumed to have an “open”-loop conformation, leads to two other conformations of the Michaelis complex, labeled inactive and active, which are probably of “closed”-loop conformations with varying loop structure and an atomic arrangement in the active site. The two closed conformations do not appear to directly interconvert but do so via the encounter complex.…”

Section: Resultssupporting

confidence: 91%

See 1 more Smart Citation

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

et al. 2016

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 91%

“…Because oxamate is a non-reactive species, the arrow labeled “chemical step” purely reflects the presumed relative reactivity between the observed conformations (cf. refs 5 and 15). …”

Section: Figurementioning

confidence: 99%

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

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…The hydrogen bonding between Arg169 and the carboxyl group is quite strong, as it populates in almost all the snapshots (99.3%) of the basin, which is consistent with the experimental results. 1 There is also a weak hydrogen bond between Thr248 and the carboxyl group, which populates in about one-quarter of the snapshots. For basin A, the substrate can be seen as rotating around its main chain by 180°.…”

Section: Resultsmentioning

confidence: 99%

“…Recent experiments have shown that conformational heterogeneity exists in the Michaelis complex of LDH, 1–3 and the propensity toward the hydride transfer reaction may vary widely among the different substates of the Michaelis complex. However, the relationship between the substates is still elusive, and the detailed structural features of the substates are unclear.…”

Section: Introductionmentioning

confidence: 99%

Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Pan

Schwartz

2015

J. Phys. Chem. B

View full text Add to dashboard Cite

It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

show abstract

“…Our analysis, by and large, concentrates on the [ 13 C 2 =O] stretch region since the [ 12 C 2 =O] stretch can somewhat couple to other oxamate vibrational modes, distorting the mode pattern. 20 …”

Section: Resultsmentioning

confidence: 99%

Mechanism of Thermal Adaptation in the Lactate Dehydrogenases

et al. 2015

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Conformational Heterogeneity within the Michaelis Complex of Lactate Dehydrogenase

Cited by 30 publications

References 27 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

Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Mechanism of Thermal Adaptation in the Lactate Dehydrogenases

Contact Info

Product

Resources

About