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Biomedical subjects

W C Kenney

Publications and source records attributed to W C Kenney.

At least 55 records · Page 3Linked to original sources
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

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1978

A novel type of covalently bound coenzyme in trimethylamine dehydrogenase.

Bacterial trimethylamine dehydrogenase contains a covalently bound yellow coenzyme, the properties of which distinguish it from all known riboflavin, pyridoxine, and pteridine derivatives. A pure dodecapeptide containing the covalently linked coenzyme has been isolated from tryptic-chymotryptic digests. Treatment with aminopeptidase M converts it to a ninhydrin-positive aminoacyl coenzyme, which has also been isolated in chromatographically pure form. The coenzyme as isolated is virtually nonfluorescent but, being extremely photolabile, it is rapidly converted on illumination to products, the predominant one being highly fluorescent. Analysis for phosphate and the influence of phosphatase treatment on electrophoretic mobility show that the coenzyme is isolated as a monophosphate, while periodate titrations prove the presence of a pentityl side chain, to which the phosphate is attached. Oxidation of the aminoacyl coenzyme with performic acid, followed by acid hydrolysis, yields 2',5'-anhydroriboflavin and some free riboflavin, products also obtained on similar treatment of FMN. These observations and the very tight binding to apoflavodoxin show that the coenzyme is a flavin derivative. The absorption and NMR spectra, however, clearly set it apart from 8alpha-substituted flavins and suggest that the coenzyme is the first representative of a new class of covalently bound flavins.

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

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.

Alcohol Oxidoreductases↗
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

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↗
PubMed1975

The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.

Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.

Animals↗