Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique

Graupe, Klaus; Trommer, Wolfgang E.; Bisswanger, Hans

doi:10.1016/0167-4838(89)90215-x

Cited by 4 publications

(4 citation statements)

References 21 publications

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“…These experiments indicated that the cysteine residue in the putative ThDP binding fold is involved in cofactor binding (). These observations are consistent with those reported earlier ( , ). The latter authors showed that the ESR spectrum of E1 modified by a spin-labeled analogue of p -hydroxymercuribenzoate is significantly changed upon addition of ThDP and Mg(II), indicating that binding ThDP·Mg(II) induces a conformational change close to an essential SH group.…”

Section: Discussionsupporting

confidence: 94%

“…Elsewhere, it was shown that E1 component resolved from E. coli PDHc is inactivated by a 3-4-fold molar excess of p-hydroxymercuribenzoate (modification of 1 cysteine residue/ monomer of E1 led to inactivation of the enzyme), yet under similar conditions, a 200-fold molar excess of DTNB or a 100-fold molar excess of iodoacetate did not affect the E1 activity. It was shown that mercurials less polar than p-hydroxymercuribenzoate inactivate the E1 component faster than p-hydroxymercuribenzoate did, indicating that hydrophobic interactions between the tested mercurials and the environment of an essential cysteine residue are responsible for the reactivity of the SH group (29).…”

Section: Discussionmentioning

confidence: 97%

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Systematic Study of the Six Cysteines of the E1 Subunit of the Pyruvate Dehydrogenase Multienzyme Complex from Escherichia coli: None Is Essential for Activity

et al. 1998

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Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

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Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 97%

Systematic Study of the Six Cysteines of the E1 Subunit of the Pyruvate Dehydrogenase Multienzyme Complex from Escherichia coli: None Is Essential for Activity

et al. 1998

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“…However, this conclusion is somewhat speculative in view of the fact that glutathionyl conjugates of BT-NE-2 have not yet been detected following incubations of DHBT-NE-1 (or BT-NE-2) and mitochondrial membranes in the presence of GSH. Covalent modification of active site cysteinyl residues of PDHC ( , ), α-KGDH (), and/or their dihydrolipoate cofactor by 2 , 7 , and possibly 15 might account for inhibition of these enzyme complexes by DHBT-NE-1.…”

Section: Discussionmentioning

confidence: 99%

Oxidative Metabolites of 5-S-Cysteinylnorepinephrine Are Irreversible Inhibitors of Mitochondrial Complex I and the α-Ketoglutarate Dehydrogenase and Pyruvate Dehydrogenase Complexes: Possible Implications for Neurodegenerative Brain Disorders

Xin

Shen

et al. 2000

Chem. Res. Toxicol.

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The major initial product of the oxidation of norepinephrine (NE) in the presence of L-cysteine is 5-S-cysteinylnorepinephrine which is then further easily oxidized to the dihydrobenzothiazine (DHBT) 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1, 4-benzothiazine-3-carboxylic acid (DHBT-NE-1). When incubated with intact rat brain mitochondria, DHBT-NE-1 evokes rapid inhibition of complex I respiration without affecting complex II respiration. DHBT-NE-1 also evokes time- and concentration-dependent irreversible inhibition of NADH-coenzyme Q(1) (CoQ(1)) reductase, the pyruvate dehydrogenase complex (PDHC), and alpha-ketoglutarate dehydrogenase (alpha-KGDH) when incubated with frozen and thawed rat brain mitochondria (mitochondrial membranes). The time dependence of the inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by DHBT-NE-1 appears to be related to its oxidation, catalyzed by an unknown component of the inner mitochondrial membrane, to electrophilic intermediates which bind covalently to active site cysteinyl residues of these enzyme complexes. The latter conclusion is based on the ability of glutathione to block inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by scavenging electrophilic intermediates, generated by the mitochondrial membrane-catalyzed oxidation of DHBT-NE-1, forming glutathionyl conjugates, several of which have been isolated and spectroscopically identified. The possible implications of these results to the degeneration of neuromelanin-pigmented noradrenergic neurons in the locus ceruleus in Parkinson's disease are discussed.

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“…ent conformational states of the enyzme, or by allosteric interaction between two identical substrate-binding sites [31]. The literature contains various possibilities to explain a nonlinear Lineweaver-Burk plot in the case of PDHC: (a) interaction between pyruvate binding sites [32]; (b) interaction between acetyl-CoA-binding sites [33]; (c) different phosphorylation sites [34], and (d) different states of the enzyme complex [35,36]. One plausible explanation for the calculated Hill coefficient of 1.63 is that PDHC in human skeletal muscle has two active sites, which stimulate each other while they bind pyruvate [29],…”

Section: Discussionmentioning

confidence: 99%

Measurement of Totally Activated Pyruvate Dehydrogenase Complex Activity in Human Muscle: Evaluation of a Useful Assay

Sperl

Trijbels²,

Ruitenbeek³

et al. 1993

Enzyme Protein

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A sensitive radiochemical method for the determination of the pyruvate dehydrogenase complex (PDHC) activity in skeletal muscle tissue, based on the decarboxylation of [1-14C]-pyruvate to 14CO(2), is described. Measurements can be carried out either in muscle homogenate or in 600-g supernatant, both obtainable from a small muscle biopsy specimen (20 mg). In addition to NAD^+, thiamine pyrophosphate and coenzyme A in the incubation mixture, a preparation of NADH:cytochrome c reductase (NADHCR) together with cytochrome c has a stimulating effect on the PDHC activity. NADHCR constitutes an oxidation system for NADH to prevent feedback inhibition. Addition of L-camitine also results in stimulation of PDHC by trapping the produced acetyl-CoA as acetylcarnitine. Special care for radioactive pyruvate, with freeze drying and storage at -20 °C under nitrogen, and determination of the purity during every PDHC assay, is required. In the presented assay a K(m) value of 0.084 mmol/l was found for pyruvate. Nonsigmoidal kinetics was found with a Hill coefficient of 1.63. With the described method, a totally Mg^2+,Ca^2+-stimulated PDHC activity is measured. Addition of a purified specific pyruvate dehydrogenase phosphatase did not yield a higher PDHC activity. Finally, comparison of total PDHC activity with [1-14C]-pyruvate oxidation rates, both measured in the supernatant prepared from fresh muscle, shows an equimolar correlation, indicating that total PDHC activity is rate limiting in the assay for the pyruvate oxidation rate. Neonatal muscle exhibits five to ten times lower PDHC activities and pyruvate oxidation rates than controls (age > 3 years).

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Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique

Cited by 4 publications

References 21 publications

Systematic Study of the Six Cysteines of the E1 Subunit of the Pyruvate Dehydrogenase Multienzyme Complex from Escherichia coli: None Is Essential for Activity

Systematic Study of the Six Cysteines of the E1 Subunit of the Pyruvate Dehydrogenase Multienzyme Complex from Escherichia coli: None Is Essential for Activity

Oxidative Metabolites of 5-S-Cysteinylnorepinephrine Are Irreversible Inhibitors of Mitochondrial Complex I and the α-Ketoglutarate Dehydrogenase and Pyruvate Dehydrogenase Complexes: Possible Implications for Neurodegenerative Brain Disorders

Measurement of Totally Activated Pyruvate Dehydrogenase Complex Activity in Human Muscle: Evaluation of a Useful Assay

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