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Luke I Szweda - One of the best experts on this subject based on the ideXlab platform.
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selective inactivation of alpha ketoglutarate Dehydrogenase and pyruvate Dehydrogenase Reaction of lipoic acid with 4 hydroxy 2 nonenal
Biochemistry, 1998Co-Authors: Kenneth M Humphries, Luke I SzwedaAbstract:Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting α-ketoglutarate Dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate Dehydrogenase (PDH), while other NADH-linked Dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in ...
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selective inactivation of alpha ketoglutarate Dehydrogenase and pyruvate Dehydrogenase Reaction of lipoic acid with 4 hydroxy 2 nonenal
Biochemistry, 1998Co-Authors: Kenneth M Humphries, Luke I SzwedaAbstract:Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate Dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate Dehydrogenase (PDH), while other NADH-linked Dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.
Dinesh Christendat - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of a novel shikimate Dehydrogenase from haemophilus influenzae
Journal of Biological Chemistry, 2005Co-Authors: Sasha A. Singh, Sergey Korolev, Olga Koroleva, T. I. Zarembinski, Frank R. Collart, Andrzej Joachimiak, Dinesh ChristendatAbstract:To date two classes of shikimate Dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate Dehydrogenase annotated as shikimate Dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ∼1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate Dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate Dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate Dehydrogenase Reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate Dehydrogenase.
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Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae
Journal of Biological Chemistry, 2005Co-Authors: Sasha A. Singh, Sergey Korolev, Olga Koroleva, T. I. Zarembinski, Frank R. Collart, Andrzej Joachimiak, Dinesh ChristendatAbstract:To date two classes of shikimate Dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate Dehydrogenase annotated as shikimate Dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is approximately 1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate Dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate Dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate Dehydrogenase Reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate Dehydrogenase.
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use of site directed mutagenesis to identify residues specific for each Reaction catalyzed by chorismate mutase prephenate Dehydrogenase from escherichia coli
Biochemistry, 1998Co-Authors: Dinesh Christendat, Vivian Saridakis, Joanne L TurnbullAbstract:Site-directed mutagenesis was performed on the bifunctional enzyme chorismate mutase-prephenate Dehydrogenase in order to identify groups important for each of the two Reactions. We selected two residues for mutagenesis, Lys37 and His131, identified previously by differential peptide mapping to be essential for activity [Christendat, D., and Turnbull, J. (1996) Biochemistry 35, 4468-4479]. Kinetic studies reveal that K37Q exhibits no mutase activity while retaining wild-type Dehydrogenase activity, verifying that Lys37 plays a key role in the mutase. By contrast His131 is not critical for the Dehydrogenase; H131A is a reasonably efficient catalyst exhibiting 10% Dehydrogenase and 30% mutase activity compared to the wild-type enzyme. Chemical modification of H131A by diethyl pyrocarbonate further inactivated the Dehydrogenase, suggesting that a different histidine is now accessible to modification. To identify this group, the protein's remaining eight histidines were changed to alanine or asparagine. A single substitution, H197N, decreased the Dehydrogenase activity by 5 orders of magnitude while full mutase activity was retained. In H197N, the Michaelis constants for prephenate and NAD+ and the mutant's elution profile from Sepharose-AMP were similar to those of wild-type enzyme, indicating that catalysis rather than substrate binding is altered. Log V for the Dehydrogenase Reaction catalyzed by H197N is pH-independent and is in contrast to wild-type enzyme, which shows a decrease in activity at low pH and pK of about 6.5. We conclude that His197 is an essential catalytic residue in the Dehydrogenase Reaction.
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identification of active site residues of chorismate mutase prephenate Dehydrogenase from escherichia coli
Biochemistry, 1996Co-Authors: Dinesh Christendat, Joanne L TurnbullAbstract:Chemical modification studies of the bifunctional enzyme chorismate mutase−prephenate Dehydrogenase and mass spectral analysis of peptide fragments containing modified residues are presented. The Reaction with diethyl pyrocarbonate (DEPC) results in the modification of several enzymic groups, including a single histidine group essential for Dehydrogenase activity and a single lysine residue essential for mutase activity. This conclusion is based on the following evidence. (1) Hydroxylamine rapidly restores Dehydrogenase activity to the DEPC-inactivated enzyme without restoring mutase activity. (2) Mutase activity is also lost upon treatment of the enzyme with trinitrobenzene sulfonate. (3) The reactivity of the Dehydrogenase to DEPC increases with pH, suggesting the participation of a group with a pKa of 7.0 in the Dehydrogenase Reaction. (4) Two peptides identified by differential peptide mapping had mass values matching those calculated for peptides comprising residues 127−135 (containing His131) and re...
Joanne L Turnbull - One of the best experts on this subject based on the ideXlab platform.
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use of site directed mutagenesis to identify residues specific for each Reaction catalyzed by chorismate mutase prephenate Dehydrogenase from escherichia coli
Biochemistry, 1998Co-Authors: Dinesh Christendat, Vivian Saridakis, Joanne L TurnbullAbstract:Site-directed mutagenesis was performed on the bifunctional enzyme chorismate mutase-prephenate Dehydrogenase in order to identify groups important for each of the two Reactions. We selected two residues for mutagenesis, Lys37 and His131, identified previously by differential peptide mapping to be essential for activity [Christendat, D., and Turnbull, J. (1996) Biochemistry 35, 4468-4479]. Kinetic studies reveal that K37Q exhibits no mutase activity while retaining wild-type Dehydrogenase activity, verifying that Lys37 plays a key role in the mutase. By contrast His131 is not critical for the Dehydrogenase; H131A is a reasonably efficient catalyst exhibiting 10% Dehydrogenase and 30% mutase activity compared to the wild-type enzyme. Chemical modification of H131A by diethyl pyrocarbonate further inactivated the Dehydrogenase, suggesting that a different histidine is now accessible to modification. To identify this group, the protein's remaining eight histidines were changed to alanine or asparagine. A single substitution, H197N, decreased the Dehydrogenase activity by 5 orders of magnitude while full mutase activity was retained. In H197N, the Michaelis constants for prephenate and NAD+ and the mutant's elution profile from Sepharose-AMP were similar to those of wild-type enzyme, indicating that catalysis rather than substrate binding is altered. Log V for the Dehydrogenase Reaction catalyzed by H197N is pH-independent and is in contrast to wild-type enzyme, which shows a decrease in activity at low pH and pK of about 6.5. We conclude that His197 is an essential catalytic residue in the Dehydrogenase Reaction.
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identification of active site residues of chorismate mutase prephenate Dehydrogenase from escherichia coli
Biochemistry, 1996Co-Authors: Dinesh Christendat, Joanne L TurnbullAbstract:Chemical modification studies of the bifunctional enzyme chorismate mutase−prephenate Dehydrogenase and mass spectral analysis of peptide fragments containing modified residues are presented. The Reaction with diethyl pyrocarbonate (DEPC) results in the modification of several enzymic groups, including a single histidine group essential for Dehydrogenase activity and a single lysine residue essential for mutase activity. This conclusion is based on the following evidence. (1) Hydroxylamine rapidly restores Dehydrogenase activity to the DEPC-inactivated enzyme without restoring mutase activity. (2) Mutase activity is also lost upon treatment of the enzyme with trinitrobenzene sulfonate. (3) The reactivity of the Dehydrogenase to DEPC increases with pH, suggesting the participation of a group with a pKa of 7.0 in the Dehydrogenase Reaction. (4) Two peptides identified by differential peptide mapping had mass values matching those calculated for peptides comprising residues 127−135 (containing His131) and re...
Kenneth M Humphries - One of the best experts on this subject based on the ideXlab platform.
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selective inactivation of alpha ketoglutarate Dehydrogenase and pyruvate Dehydrogenase Reaction of lipoic acid with 4 hydroxy 2 nonenal
Biochemistry, 1998Co-Authors: Kenneth M Humphries, Luke I SzwedaAbstract:Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting α-ketoglutarate Dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate Dehydrogenase (PDH), while other NADH-linked Dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in ...
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selective inactivation of alpha ketoglutarate Dehydrogenase and pyruvate Dehydrogenase Reaction of lipoic acid with 4 hydroxy 2 nonenal
Biochemistry, 1998Co-Authors: Kenneth M Humphries, Luke I SzwedaAbstract:Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate Dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate Dehydrogenase (PDH), while other NADH-linked Dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.
Victoria I Bunik - One of the best experts on this subject based on the ideXlab platform.
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advantages of formate Dehydrogenase Reaction for efficient nad quantification in biological samples
Analytical Biochemistry, 2020Co-Authors: Artem V Artiukhov, V I Tishkov, A A Pometun, Sofia A Zubanova, Victoria I BunikAbstract:The medical significance of NAD+-dependent metabolic regulation acquires increasing attention, demanding rapid and clinically feasible quantification of NAD+ in complex biological samples. Here we describe the usage of formate Dehydrogenase for a straightforward and highly specific fluorometric assay of NAD+ in tissue extracts, not requiring chromatographic separation of nucleotides. The assay employs the irreversible Reaction of formate oxidation coupled to NAD+ reduction, catalyzed by the enzyme which has high affinity and specificity to NAD+, and is stable under a variety of conditions. The assay reliably quantifies NAD+ in the methanol extracts of the rat brain cortex and mitochondria.
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regulation of malate Dehydrogenases and glutamate Dehydrogenase of mammalian brain by thiamine in vitro and in vivo
Biochemistry, 2020Co-Authors: O A Mezhenska, Artem V Artiukhov, Victoria I Bunik, V A Aleshin, Thilo KaehneAbstract:To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate Dehydrogenase (GDH) and isoenzymes of malate Dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate Dehydrogenase Reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elu-tion by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with “phosphoprotein”, “acetylation”, and “methylation”. The relationship between thiamine and the post-translational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.
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specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate Dehydrogenase Reaction is essential for metabolism and viability of glioblastoma cells
Oncotarget, 2015Co-Authors: Victoria I Bunik, Artem V Artiukhov, Alisdair R Fernie, A V Kazantsev, Renata L S Goncalves, Danilo M Daloso, Henry Oppermann, Elena Kulakovskaya, N V Lukashev, Martin D BrandAbstract:The pyruvate Dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid Dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate Dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.
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metabolic control exerted by the 2 oxoglutarate Dehydrogenase Reaction a cross kingdom comparison of the crossroad between energy production and nitrogen assimilation
Biochemical Journal, 2009Co-Authors: Victoria I Bunik, Alisdair R FernieAbstract:Mechanism-based inhibitors and both forward and reverse genetics have proved to be essential tools in revealing roles for specific enzymatic processes in cellular function. Here, we review experimental studies aimed at assessing the impact of OG (2-oxoglutarate) oxidative decarboxylation on basic cellular activities in a number of biological systems. After summarizing the catalytic and regulatory properties of the OGDHC (OG Dehydrogenase complex), we describe the evidence that has been accrued on its cellular role. We demonstrate an essential role of this enzyme in metabolic control in a wide range of organisms. Targeting this enzyme in different cells and tissues, mainly by its specific inhibitors, effects changes in a number of basic functions, such as mitochondrial potential, tissue respiration, ROS (reactive oxygen species) production, nitrogen metabolism, glutamate signalling and survival, supporting the notion that the evolutionary conserved Reaction of OG degradation is required for metabolic adaptation. In particular, regulation of OGDHC under stress conditions may be essential to overcome glutamate excitotoxicity in neurons or affect the wound response in plants. Thus, apart from its role in producing energy, the flux through OGDHC significantly affects nitrogen assimilation and amino acid metabolism, whereas the side Reactions of OGDHC, such as ROS production and the carboligase Reaction, have biological functions in signalling and glyoxylate utilization. Our current view on the role of OGDHC Reaction in various processes within complex biological systems allows us a far greater fundamental understanding of metabolic regulation and also opens up new opportunities for us to address both biotechnological and medical challenges.