The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [L-(+)-lactate dehydrogenase].
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ABSTRACT:
SUBMITTER: Genet R
PROVIDER: S-EPMC1131127 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
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Project description:L-Lactate dehydrogenase (L-LDH) from Saccharomyces cerevisiae and L-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis are both flavocytochromes b2. The kinetic properties of these enzymes have been compared using steady-state kinetic methods. The most striking difference between the two enzymes is found by comparing their substrate specificities. L-LDH and L-MDH have mutually exclusive primary substrates, i.e. the substrate for one enzyme is a potent competitive inhibitor for the other. Molecular-modelling studies on the known three-dimensional structure of S. cerevisiae L-LDH suggest that this enzyme is unable to catalyse the oxidation of L-mandelate because productive binding is impeded by steric interference, particularly between the side chain of Leu-230 and the phenyl ring of mandelate. Another major difference between L-LDH and L-MDH lies in the rate-determining step. For S. cerevisiae L-LDH, the major rate-determining step is proton abstraction at C-2 of lactate, as previously shown by the 2H kinetic-isotope effect. However, in R. graminis L-MDH the kinetic-isotope effect seen with DL-[2-2H]mandelate is only 1.1 +/- 0.1, clearly showing that proton abstraction at C-2 of mandelate is not rate-limiting. The fact that the rate-determining step is different indicates that the transition states in each of these enzymes must also be different.
| S-EPMC1132387 | biostudies-other
Project description:Flavocytochrome b2 from Saccharomyces cerevisiae acts physiologically as an L-lactate dehydrogenase. Although L-lactate is its primary substrate, the enzyme is also able to utilize a variety of other (S)-2-hydroxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues required for catalysis. However, the residues Ala-198 and Leu-230, found in the X-ray-crystal structure to be in contact with the substrate methyl group, are not well conserved. We propose that the interaction between these residues and a prospective substrate molecule has a significant effect on the substrate specificity of the enzyme. In an attempt to modify the specificity in favour of larger substrates, three mutant enzymes have been produced: A198G, L230A and the double mutant A198G/L230A. As a means of quantifying the overall kinetic effect of a mutation, substrate-specificity profiles were produced from steady-state experiments with (S)-2-hydroxy acids of increasing chain length, through which the catalytic efficiency of each mutant enzyme with each substrate could be compared with the corresponding wild-type efficiency. The Ala-198-->Gly mutation had little influence on substrate specificity and caused a general decrease in enzyme efficiency. However, the Leu-230-->Ala mutation caused the selectivity for 2-hydroxyoctanoate over lactate to increase by a factor of 80.
| S-EPMC1137062 | biostudies-other
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.
| S-EPMC1948838 | biostudies-literature
Project description:Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.
| S-EPMC1219563 | biostudies-other
Project description:His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.
| S-EPMC2526232 | biostudies-literature
Project description:Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.
| S-EPMC2794998 | biostudies-literature
Project description:Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.
| S-EPMC8560939 | biostudies-literature
Project description:Effective glucose diet: We report the development and activity of glucose-conjugated LDH-A inhibitors designed for dual targeting of the Warburg effect (elevated glucose uptake and glycolysis) in cancer cells. Glycoconjugation could be applied to inhibitors of many enzymes involved in glycolysis or tumor metabolism.
| S-EPMC3919968 | biostudies-literature
Project description:Cancer is one of the main causes of mortality in the world. Many cancer cells produce ATP through high-level lactic acid fermentation catalyzed by lactate dehydrogenase (LDH), which converts pyruvic acid to lactic acid. LDH plays a dominant role in the Warburg effect, wherein aerobic glycolysis is favored over oxidative phosphorylation. Due to the high lactic acid production level in cancer cells, LDH-targeting could be a potential cancer treatment strategy. A few approaches, such as drug treatment, reportedly inhibited LDH activity. In this study, we describe new 1,3-benzodioxole derivatives that might be potential small molecule candidates for LDHA inhibition. The synthesis was carried out by trans-esterification between aryl ester and alcohol groups from piperonyl alcohol. Compounds 2 and 10 exhibited a selective LDHA IC50 value of 13.63 µM and 47.2 µM, respectively. Whereas only compound 10 showed significant cytotoxicity in several lines of cancer cells, especially in human pancreatic cancer PANC-1 cells. These synthesized compounds possess 2 aromatic rings and -CF3 moiety, which expectedly contributes to LDHA inhibition. The presented products have the potential to become a promising LDHA inhibitor drug candidate.
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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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