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T P Singer

Publications and source records attributed to T P Singer.

At least 127 records · Page 7Linked to original sources
PubMed1977

Identification of the iron-sulfur center in trimethylamine dehydrogenase.

Trimethylamine dehydrogenase [trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7] from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*). The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure. Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type. Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes [Fe4S*4(S2-o-xylyl)2]n2n- and [Fe4S*4(SC6H4OMe)4]2-, which were identified by absorption spectra. These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster [Fe4S*4(S-Cys)4].

Bacteria↗
PubMed1976

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.

Amino Acids↗
PubMed1976

Structural elucidation and properties of 8alpha-(N1-histidyl)riboflavin: the flavin component of thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase.

In addition to 8alpha-(N3-histidyl)riboflavin, 8alpha-(N1-histidyl)riboflavin is also formed during the reaction of Nalpha-blocked histidine with 8alpha-bromotetraacetylriboflavin in a yield of 20-25% of the total histidylflavin fraction. The properties of 8alpha-(N1-histidyl)riboflavin are inditical with those of the histidylflavin isolated from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase but differ from those of 8alpha-(N3-histidyl)riboflavin. These properties include pKa of fluorescence quenching, electrophoretic mobility at pH 5.0, stability to storage, and reduction by NaBH4. Proof for 8alpha substitution is shown by the electron paramagnetic resonance and electron-nuclear double resonance spectra of the cationic semiquinone form, as well as by the proton magnetic resonance spectrum of the oxidized form. The site of histidine substitution by the 8alpha-methylene of the flavin moiety was shown by methylation of the imidazole ring with methyl iodide, cleavage of the methylhistidine-flavin bond by acid hydrolysis at 150 degrees C, and identification of the methylhistidine isomer by electrophoresis. 3-Methylhistidine is the product from the N1-histidylflavin isomer, while 1-methylhistidine is produced from the N3 isomer. The flavin product from reductive Zn cleavage of either isomer has been identified as riboflavin. The compound obtained on acid treatment of 8alpha-(N3-histidyl)riboflavin (previously thought to be the N1 isomer) differs from the parent compound only in the ribityl side chain, since chemical degradation studies show 1-methylhistidine as a product and a flavin product which differs from riboflavin only in mobility in thin-layer chromatography, but not in absorption, fluorescence, and electron paramagnetic resonance spectral properties. Proof that acid modification involves only the ribityl chain has come from the observations that alkaline irradiation of this flavin yields lumiflavin, that the proton magnetic resonance spectrum of the compound differs from that of riboflavin in the region of the ribityl proton resonance, and that its periodate titer is lower than that of authentic riboflavin. The identity of 8alpha-(N1-histidyl)riboflavin with the histidylflavin from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase shows that both isomeric forms of 8alpha-histidylflavin occur in nature.

Alcohol Oxidoreductases↗
PubMed1976

Transport of riboflavin into yeast cells.

Riboflavin-requiring mutants of Saccharomyces cerevisiae are able to transport 14C-labeled riboflavin into the cell, although no significant transport is seen in commercial yeast or in the parent strain from which the mutants were derived. Transport activity is greatest in the early to mid-log phase of anaerobic growth and declines sharply in the late log phase. In aerobically grown cells activity is substantially lower at all stages of growth. In the assay devised for its measurement, transport activity shows a sharp pH optimum at pH 7.5, a strong temperature dependence (EA = 23,100 cal/mol), and saturation kinetics with respect to riboflavin (Km = 15 muM), characteristics consistent with a carrier-mediated mechanism. Monovalent inorganic cations, particularly K+ and Rb+, stimulate riboflavin uptake, while certain organic cations are inhibitory. Besides riboflavin only 7-methylriboflavin, 8-methylriboflavin, and 5-deazaflavin have been found to serve as substrates, while lumiflavin, tetraacetylriboflavin, and N10-[4'-carboxybutyl]-7,8-dimethylisoalloxazine do not, although a number of flavin analogs in which the ribityl side chain is modified are good competitive inhibitors of riboflavin uptake. Compounds resembling the ribityl side chain, such as sugars and sugar alcohols, do not inhibit. An apparent inhibition of uptake by D-glucose, D-mannose, and D-fructose, which develops in the course of assay, proved to result from stimulation of an opposing process, the release of riboflavin from the cells.

Anaerobiosis↗
PubMed1976

Localization of the substrate and oxalacetate binding site of succinate dehydrogenase.

Succinate dehydrogenase is composed of two subunits, one of molecular weight 70,000, containing FAD in covalent linkage to a histidyl residue of the polypeptide chain, the other subunit of molecular weight 30,000. The fact that substrate, substrate analogs, and oxalacetate prevent inactivation of the enzyme by thiol-specific agents indicates that a thiol group must be present in close proximity to the flavin. Comparison of the incorporation of radioactivity into each subunit in the presence and absence of succinate or malonate shows that both substrate and competitive inhibitors protect a sulfhydryl group of the 70,000-molecular weight subunit. This indicates that a thiol group of the flavoprotein subunit is part of the active site. Similar investigations using oxalacetate as a protecting agent indicate that the tight binding of oxalacetate to the deactivated enzyme also occurs in the flavoprotein subunit, and may involve the same thiol group which is protected by succinate from alkylation by N-ethylmaleimide. It is clear, therefore, that not only the flavin site but also an essential thiol residue are located in the 70,000-molecular weight subunit. A second thiol group, located in the 30,000-molecular weight subunit, also binds N-ethylmaleimide covalently under similar conditions, without being part of the active site. Succinate, malonate, and oxalacetate do not influence the binding of this inhibitor to the thiol group of the lower molecular weight subunit. Using maleimide derivatives of nitroxide-type spin labels, it has been possible to demonstrate the presence of two types of thiol groups in the enzyme which form covalent derivatives with the spin probe. When the enzyme is treated with an equimolar quantity of the spin probe, a largely isotropic electron spin resonance spectrum is obtained, indicating a high probe mobility. When this site is first blocked by treating the enzyme with an equimolar quantity of N-ethylmaleimide, followed by an equimolar amount of spin label, the label is strongly immobilized with a splitting of 64 gauss. It is suggested that the sulfhydryl group which is involved in the immobilized species is at the active site.

Binding Sites↗
PubMed1976

The structure of the covalent adduct formed by the interaction of 3-dimethylamino-1-propyne and the flavine of mitochondrial amine oxidase.

3-Dimethylamino-1-propyne irreversibly inactivates mitochondrial monoamine oxidase from bovine liver. The inactivation results in the loss of absorption in the 450-500-nm region of the flavine spectrum and a concomitant increase in absorbance at 410 nm. For the enzyme-bound adduct epsilon410 = 28000. The spectral properties of the adduct of the liver enzyme with 3-dimethylamino-1-propyne are similar to those observed when the pig kidney enzyme is inactivated with pargyline (Chuang et al. (1974), J. Biol. Chem. 249, 2381). From a proteolytic digest of the enzyme inactivated with labeled inhibitor a flavine peptide has been isolated which contains 1 mol of inactivator/mol of flavine. The chemical and spectral properties of the adduct are those of compounds containing the structure --N--CH==CH--CH==N+ less than. It was concluded that the flavine-inhibtor adduct is a N-5 substituted dihydroflavine and its structure has been determined.

Alkynes↗
PubMed1975

Piericiden A sensitivity, site 1 phosphorylation, and reduced nicotinamide adenine dinucleotide dehydrogenase during iron-limited growth of Candida utilis.

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th

Aerobiosis↗
PubMed1975

Iron-sulfur components of succinate dehydrogenase: stoichiometry and kinetic behavior in activated preparations.

Extensively or completely activated preparations of beef heart succinate dehydrogenase have been investigated by electron paramagnetic resonance (EPR) techniques at 6 to 97 K. Reductive titrations with dithionite and rapid kinetic studies were performed with various types of soluble and membrane-bound preparations of the enzyme. The following components were detected and their behavior analyzed: a free radical, presumably arising from the covalently bound flavin on reduction, two iron-sulfur centers of the ferredoxin type, the signals of which appear on reduction, and a highpotential iron-sulfur component, detectable in the oxidized state. The high-potential component was only detected in complex II and inner-membrane preparations. This component and one of the ferredoxin-type centers were present in amounts close to stoichiometric with the flavin and were reduced by substrate. The other ferredoxin-type center was present in amounts between 0.1 and 0.5 times that of the flavin and was reduced only by dithionite. Of the components reduced by succinate, however, only a fraction (up to 50% of the high-potential iron-sulfur center and 40-60% of the ferredoxin-type iron-sulfur center) was reduced within the turnover time of the enzymes; In complex II not more than about 10% of the flavin appeared in the semiquinone form at any time. Soluble, purified preparations behaved similarly except that the high-potential component was nearly or completely absent and extensive accumulation of the free radical occurred (up to 70 to 80% of the flavin) in titration and kinetic experiments. No significant difference was observed between the rates of semiquinone formation and the reduction of the ferredoxin-type or high-potential centers by the substrate. Also no qualitative differences in the properties studied in this work became apparent between prepatations containing 4 or 8 iron atoms, respectively.

Animals↗