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T P Singer

Publications and source records attributed to T P Singer.

At least 91 records · Page 5Linked to original sources
PubMed1983

Resonance Raman studies of beef heart aconitase and a bacterial hydrogenase.

The resonance Raman (RR) spectra of beef heart aconitase and of an air-stable hydrogenase from Desulfuvibrio desulfuricans, as isolated, are characteristic of 3Fe centers. Activation of aconitase by Fe(II) addition converts the RR spectrum to one characteristic of [4Fe-4S]2+ clusters. Analytical data on aconitase, as isolated, confirms the recent finding (Beinert, H., Emptage, M. H., Dreyer, J.-L., Scott, R. A., Hahn, J. E., Hodgson, K. O., and Thomson, A. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 393-396) of a [3Fe-4S] stoichiometry. The RR spectra of 3Fe centers from aconitase, and the hydrogenase, as well as from several bacterial ferredoxins, conform to the pattern expected for a cube-derived [3Fe-4S] cluster. Perceptible differences are observed among the spectra, which can be explained in terms of differences among the terminal ligands, perhaps limited to their conformations. In the case of aconitase and hydrogenase, frequency shifts suggest additional alterations in the terminal Fe-S bond angles and/or slight differences in core geometry.

Aconitate Hydratase↗
PubMed1983

Inactivation of the 2-ketoglutarate and pyruvate dehydrogenase complexes of beef heart by branched chain keto acids.

Incubation of 2-ketoglutarate dehydrogenase complex with 2-ketoisovalerate, 2-keto-4-methylvalerate, or 2-keto-3-methylvalerate leads to the appearance of a lag phase and of a progressive loss of activity in subsequent measurements of the initial rate of oxidation of 2-ketoglutarate. In the case of 2-ketoisovalerate these effects are shown to be due to the formation of an isobutyryllipoate derivative of the enzyme, as a result of the very slow oxidation of 2-ketoisovalerate by the enzyme complex (Vmax congruent to 0.15% of that for 2-ketoglutarate). Incubation of the enzyme complex with 2-keto[14C]isovalerate or 2-keto[14C]glutarate results in comparable incorporation of radioactivity, amounting to 3.5 to 5.3 nmol of isobutyryl or succinyl residues per mg of protein in the complex. Isobutyryl residues are also incorporated in the enzyme during the simultaneous oxidation of both of these substrates. During the early phase of incubation of the complex with 2-ketoisovalerate the incorporation of isobutyryl residues is much faster than the loss of enzyme activity. This observation seems to support the suggestion that each 2-ketoglutarate decarboxylase subunit of the complex may catalyze the succinylation of more than one lipoate succinyltransferase subunit. Results are also presented showing the inactivation of pyruvate dehydrogenase complex on preincubation with 2-ketoisovalerate and of 2-ketoglutarate dehydrogenase complex with methylenecyclopropylpyruvate, the keto acid corresponding to the toxic amino acid hypoglycin. The relevance of covalent modifications of the two keto acid dehydrogenase complexes to the pathological manifestations of maple syrup urine disease are discussed.

Animals↗
PubMed1982

Simplified isolation and molecular composition of NADH dehydrogenase of the respiratory chain.

A simplified procedure for the isolation of NADH dehydrogenase from the inner membrane of ox heart mitochondria is presented which permits relatively rapid preparation of the enzyme in a more stable form than that afforded by published methods. The protein thus isolated displays more than eight different subunits in gel electrophoresis under denaturing conditions, three of which are also present in the "low-molecular-weight form' of the enzyme prepared under more drastic conditions. Complex I contains several subunits, mostly of low molecular weight, not seen in soluble purified NADH dehydrogenase. It is suggested that some of these may be 'binding peptides' necessary in linking NADH dehydrogenase to ubiquinone reduction, analogously to the role of small peptides in linking succinate dehydrogenase to ubiquinone. The dehydrogenase isolated by the rapid method contains equimolar amounts of non-haem iron and labile sulphur, but on further manipulation non-haem iron (but no labile sulphur) is lost, resulting in ratios of S/Fe in excess of unity, as previously reported for preparations isolated by longer procedures.

Animals↗
PubMed1981

Relationship of the oxidation state of the iron-sulfur cluster of aconitase to activity and substrate binding.

It is known that aconitase from mammalian mitochondria is only partially active as isolated but may be activated by incubation with iron, ascorbate, and a thiol, or with dithionite. It has been suggested that the added Fe in the activation mixture is essential for activation and that it is incorporated in the enzyme [Villafranca, J. J., & Mildvan, A. S. (1971) J. Biol. Chem. 246, 772-779; Gawron, O., Waheed, A., Glaid, A. J., & Jaklitsch, A. (1974) Biochem. J. 139, 709-714]. However, it is shown in this paper that, when the enzyme has a full complement of 3Fe and 3S, full activation is reached coulometrically, without iron or other chemical reducing agents. It is clear, therefore, that the role of activators is to reduce the iron--sulfur cluster of the enzyme. The appearance of catalytic activity on reduction of the cluster shows a pronounced lag, as does the decay of activity after reoxidizing the cluster. This suggests that catalytic activity requires a conformational change in the protein which is initiated by reduction of the cluster and that, following reoxidation, activity disappears only after the inactive conformation is assumed. Citrate and the competitive inhibitor trans-aconitate are bound to a comparable extent to the active and inactive forms, but only the active form can bind 1-hydroxy-2-nitro-1,3-propanedicarboxylic acid, a transition-state analogue. This is interpreted to show that in the inactive state aconitase cannot enter the conformation it assumes in the transition state during catalysis.

Aconitate Hydratase↗
PubMed1981

8 alpha-(O-Tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase.

8 alpha-(O-Tyrosyl)riboflavin has been synthesized by condensation of the copper complex of L-tyrosine with 8 alpha-bromotetraacetylriboflavin. The structure of this synthetic product was proven by absorption and 1H NMR spectroscopy and by chemical degradation, which yielded 1 mol of tyrosine per mol of flavin. The synthetic compound comigrated wtih the (aminoacyl)riboflavin isolated from the p-cresol methylhydroxylase of Pseudomonas putida, and both showed identical absorption and fluorescence spectral properties. 8 alpha-(O-Tyrosyl)riboflavin as well as the flavin-containing decapeptide from p-cresol methylhydroxylase undergoes reductive cleavage to form riboflavin and FAD, respectively, on anaerobic treatment with dithionite. In contrast, the native enzyme, on reduction with dithionite, yields a reduced flavin via a red (anionic) flavosemiquinone intermediate, which remains covalently bound to the protein even under denaturing conditions. 8 alpha-(O-Tyrosyl)riboflavin bound to apoflavodoxin is also not cleaved on reduction with dithionite, but, instead, a blue (neutral) semiquinone of tyrosylriboflavin is generated, which is resistant to further reduction with dithionite. Three p-cresol methylhydroxylases, isolated from different strains of Pseudomonas putida, differing in molecular weight and Km values for substrates, contain the same peptide at the flavin site. These data provide definitive proof for the existence of 8 alpha-(O-tyrosyl)riboflavin in nature.

Cresols↗
PubMed1981

Structural identification of iron-sulfur clusters of the respiratory chain-linked NADH dehydrogenase.

The structural nature of the iron-sulfur clusters of NADH dehydrogenase from beef heart mitochondria has been studied by the cluster extrusion technique. Enzyme samples were unfolded anaerobically in 80% (v/v) hexamethylphosphoramide/aqueous buffer in the presence of o-xylyl-alpha,alpha'-dithiol as the displacing agent and the extruded clusters were then reacted with p-trifluoromethylbenzenethiol and analyzed by Fourier transform 19F NMR at 339 MHz. Whenever extrusion was nearly complete, both binuclear and tetranuclear clusters were found at a mole ratio of approximately 2:1. Thus, the dehydrogenase, with 16 g atoms of non-heme iron present/mol of FMN, contains most likely four [2Fe-2S] and two [4Fe-4S] clusters. Because the enzyme contains four or, at the most five, EPR-detectable iron-sulfur centers, it appears that one or more of the clusters are EPR-silent.

Anaerobiosis↗
PubMed1981

Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II.

Oxathiin carboxanilides are systemic fungicides that inhibit the oxidation of succinate by interrupting electron transport between succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] and coenzyme Q. Kinetic and electron paramagnetic resonance studies have established that the specific binding site of carboxanilides and of thenoyltrifluoroacetone responsible for the inhibition is the same. Although the binding of carboxanilides to membrane preparations of the dehydrogenase is very tight (Ki = 0.01-0.1 microM), it is noncovalent. Identification of the membrane component(s) to which specific binding occurs has therefore required the introduction of a photoaffinity label onto the carboxanilide molecule. By using [G-3H]3'-azido-5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide, it was found, in accord with earlier data with other carboxanilides, that unresolved complex II specifically binds about 0.6 mol of the inhibitor per mol of succinate dehydrogenase in equilibrium dialysis experiments. The resolved components of the complex, succinate dehydrogenase and the two binding peptides CII-3 and CII-4, failed to bind the inhibitor; however, when these were recombined with reconstitution of coenzyme Q reductase activity, the initial binding titer was restored. Azidocarboxanilide-inhibited complex II was irradiated to generate covalent linkages with the binding site, and the components of the complex were separated on polyacrylamide gel. Most of the specifically bound inhibitor was found in the low molecular weight binding peptides and phospholipids.

Anilides↗
PubMed1980

Chemical synthesis and some properties of 6-substituted flavins.

A number of derivatives of riboflavin and of 3-methyllumiflavin substituted in the 6 position have been synthesized starting with 6-nitro flavins, reduction to the 6-amino flavin, and diazotization, followed by reaction with the appropriate nucleophile. The absorption spectra, oxidation-reduction potentials, and the electron spin resonance spectra of the radical cationic forms of several of these synthetic compounds have been determined, including 6-S-cysteinyl-3-methyllumiflavin and 6-S-cysteinylriboflavin. The latter has been shown to be identical with the dephosphorylated form of the aminoacyl flavin isolated from trimethylamine dehydrogenase [Steenkamp, D. J., Kenney, W. C. & Singer, T. P. (1978) J. Biol. Chem. 253, 2812-2817; Steenkamp, D. J., McIntire, W., & Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824] in regard to absorption specturm, photochemical properties, and mobility in high-voltage electrophoresis and in thin-layer chromatography. An unusually pronounced interaction between the amino group and the isoalloxazine ring system was deduced from the absorption spectra of 6-amino-3-methyllumiflavin and 6-aminoriboflavin.

Electron Spin Resonance Spectroscopy↗
PubMed1979

Transport and binding of riboflavin by Bacillus subtilis.

Riboflavine uptake and membrane-associated riboflavin-binding activity has been investigated in Bacillus subtilis. Riboflavin uptake proceeds via a system whose general properties are indicative of a carrier-mediated process: it is inhibited by substrate analogues, exhibits saturation kinetics, and is temperature-dependent. The organism concentrates riboflavin primarily as the phosphorylated cofactors FMN and FAD. Energy is required for uptake but whether the energy demand is required for both uptake and phosphorylation or only for the phosphorylation step is not known. Membrane-associated binding activity for riboflavin has also been demonstrated in membrane vesicles prepared from B. subtilis, and the binding component can be "solubilized" with Triton X-100. Evidence supporting the function of the binding component in riboflavin uptake by the intact cells includes the following. (i) Riboflavin analogues inhibit binding and uptake to nearly the same extent and with similar specificity of action. (ii) The KD for riboflavin-binding and the Km for uptake are in the same range. Similarly the Ki determined for the inhibitory analogue 5-deazariboflavin in the uptake assay and the KD for its interaction with the riboflavin-binding component of membrane vesicles are in the same range. (iii) Uptake in cells and binding in vesicles vary in the same direction with differences in growth conditions.

Bacillus subtilis↗
PubMed1979

The high potential iron-sulfur cluster of aconitase is a binuclear iron-sulfur cluster.

It has been reported (Ruzicka, F.J., and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517) that aconitase in the oxidized state, as isolated, shows an electron paramagnetic resonance signal centered at g = 2.01, typical of high potential iron-sulfur proteins. Since the magnetic state corresponding to this signal has thus far only been found in tetranuclear iron-sulfur clusters in model compounds and proteins, it could be expected that aconitase also contains a [4Fe-4S] cluster. We show here that core extrusion, in the presence of hexamethylphosphoramide and o-xylyl-alpha,alpha'-dithiol and subsequent ligand exchange with p-trifluoromethylbenzenethiol yield absorption spectra typical of binuclear iron-sulfur clusters. According to the absorbance measured, the concentration of the extruded [2Fe-2S] cluster quantitatively accounts for the iron-sulfur content of the preparations examined. Preliminary studies of the 19F nuclear magnetic resonance spectrum obtained on extrusion with p-trifluoromethylbenzenethiol confirm the presence of a binuclear cluster in aconitase.

Aconitate Hydratase↗