Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding.
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ABSTRACT:
SUBMITTER: Holbrook JJ
PROVIDER: S-EPMC1179599 | biostudies-other | 1970 Nov
REPOSITORIES: biostudies-other
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Project description:Recent experimental studies suggest that lactate dehydrogenase (LDH) binds its substrate via the formation of a LDH/NADH.substrate encounter complex through a select-fit mechanism, whereby only a minority population of LDH/NADH is binding-competent. In this study, we perform molecular dynamics calculations to explore the variations in structure accessible to the binary complex with a focus on identifying structures that seem likely to be binding-competent and which are in accord with the known experimental characterization of forming binding-competent species. We find that LDH/NADH samples quite a range of protein conformations within our 2.148 ns calculations, some of which yield quite facile access of solvent to the active site. The results suggest that the mobile loop of LDH is perhaps just partially open in these conformations and that multiple open conformations, yielding multiple binding pathways, are likely. These open conformations do not require large-scale unfolding/melting of the binary complex. Rather, open versus closed conformations are due to subtle protein and water rearrangements. Nevertheless, the large heat capacity change observed between binding-competent and binding-incompetent can be explained by changes in solvation and an internal rearrangement of hydrogen bonds. We speculate that such a strategy for binding may be necessary to get a ligand efficiently to a binding pocket that is located fairly deep within the protein's interior.
| S-EPMC1948035 | biostudies-literature
Project description:Employing temperature-jump relaxation spectroscopy, we investigate the kinetics and thermodynamics of the formation of a very early ternary binding intermediate formed when lactate dehydrogenase (LDH) binds a substrate mimic on its way to forming the productive LDH/NADH.substrate Michaelis complex. Temperature-jump scans show two distinct submillisecond processes are involved in the formation of this ternary binding intermediate, called the encounter complex here. The on-rate of the formation of the encounter complex from LDH/NADH with oxamate (a substrate mimic) is determined as a function of temperature and in the presence of small concentrations of a protein destabilizer (urea) and protein stabilizer (TMAO). It shows a strong temperature dependence with inverse Arrhenius behavior and a temperature-dependent enthalpy (heat capacity of 610 +/- 84 cal/Mol K), is slowed in the presence of TMAO and speeded up in the presence of urea. These results suggest that LDH/NADH occupies a range of conformations, some competent to bind substrate (open structure; a minority population) and others noncompetent (closed), in fast equilibrium with each other in accord with a select fit model of binding. From the thermodynamic results, the two species differ in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and/or increases in the solvation of the protein structure. The binding-competent species can bind ligand at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed to solvent in these species. This would be in contrast to the putative closed structure where the binding pocket resides deep within the protein interior.
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Project description:Adding the two residues comprising the conserved proton-transfer network of Class 2 dihydroorotate dehydrogenase (DHOD) to the Cys130Ser Class 1A DHOD did not restore the function of the active site base or rapid flavin reduction. Studies of triple, double, and single mutant Class 1A enzymes showed that the network actually prevents cysteine from acting as a base and that the network residues act independently. Our data show that residue 71 is an important determinant of ligand binding specificity. The Leu71Phe mutation tightens dihydrooroate binding but weakens the binding of benzoate inhibitors of Class 1A enzymes.
| S-EPMC3077728 | biostudies-literature
Project description:BackgroundCorynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032.ResultsHere, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer.ConclusionsCg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.
| S-EPMC3022706 | biostudies-literature
Project description:Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer.
2010-12-02 | GSE25704 | GEO
Project description:SARS-CoV-2 (COVID-19) patients who develop acute respiratory distress syndrome (ARDS) can suffer acute lung injury, or even death. Early identification of severe disease is essential in order to control COVID-19 and improve prognosis. Oxidative stress (OS) appears to play an important role in COVID-19 pathogenesis; we therefore conceived a study of the potential discriminative ability of serum biomarkers in patients with ARDS and those with mild to moderate disease (non-ARDS). 60 subjects were enrolled in a single-centre, prospective cohort study of consecutively admitted patients: 29 ARDS/31 non-ARDS. Blood samples were drawn and marker levels analysed by spectrophotometry and immunoassay techniques. C-reactive protein (CRP), lactate dehydrogenase (LDH), and ferritin were significantly higher in ARDS versus non-ARDS cases at hospital admission. Leukocytes, LDH, ferritin, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) were also significantly elevated in ARDS compared to non-ARDS patients during the hospital stay. Total thiol (TT) was found to be significantly lower in ARDS. Conversely, D-dimer, matrix metalloproteinase-9 (MMP-9) and advanced glycosylated end products (AGE) were elevated. Leukocytes, LDH, CRP, ferritin and IL-6 were found to be significantly higher in non-survivors. However, lymphocyte, tumour necrosis factor beta (TGF-β), and TT were lower. In summary, our results support the potential value of TT, ferritin and LDH as prognostic biomarkers for ARDS development in COVID-19 patients, distinguishing non-ARDS from ARDS (AUCs = 0.92; 0.91; 0.89) in a fast and cost-effective manner. These oxidative/inflammatory parameters appear to play an important role in COVID-19 monitoring and can be used in the clinical management of patients.
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Project description:Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum.
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Project description:Substrate channeling studies have frequently failed to provide conclusive results due to poor understanding of this subtle phenomenon. We analyzed the mechanism of NADH-channeling from D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to L-lactate Dehydrogenase (LDH) using enzymes from different cells. Enzyme kinetics studies showed that LDH activity with free NADH and GAPDH-NADH complex always take place in parallel. The channeling is observed only in assays that mimic cytosolic conditions where free NADH concentration is negligible and the GAPDH-NADH complex is dominant. Molecular dynamics and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at the limiting NADH concentrations. Surface calculations showed that positive electric field between the NAD(H) binding sites on LDH and GAPDH tetramers can merge in the LDH-GAPDH complex. NAD(H)-channeling within the LDH-GAPDH complex can be an extension of NAD(H)-channeling within each tetramer. In the case of a transient LDH-(GAPDH-NADH) complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between the off-rates for the LDH-(GAPDH-NADH) complex and the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.
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Project description:Kinases and phosphatases are major players in a variety of cellular events, including cell signaling. Aberrant activity or mutations in kinases and phosphatases can lead to diseases such as cancer, diabetes, and Alzheimer's. Compared to kinases, phosphatases are understudied; this is partly a result of the limited methods for identifying substrates. As a solution, we developed a proteomics-based method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) that previously identified substrates of Ser/Thr phosphatases using small molecule inhibitors. Here, for the first time, K-BIPS was applied to identify substrates of a tyrosine phosphatase, protein tyrosine phosphatase 1B (PTP1B), under siRNA knockdown conditions. Eight possible substrates of PTP1B were discovered in HEK293 cells, including the known substrate pyruvate kinase. In addition, l-lactate dehydrogenase (LDHA) was validated as a novel PTP1B substrate. With the ability to use knockdown conditions with Ser/Thr or Tyr phosphatases, K-BIPS represents a general discovery tool to explore phosphatases biology by identifying unanticipated substrates.
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Project description:Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD(+) (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD(+) by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD(+), and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation.
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