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T Toraya

Publications and source records attributed to T Toraya.

At least 73 records · Page 4Linked to original sources
PubMed1980

In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase.

The catalytic properties of coenzyme B12-dependent glycerol dehydratase and diol dehydratase were studied in situ with Klebsiella pneumoniae cells permeabilized by toluene treatment, since the in situ enzymes approximate the in vivo conditions of the enzymes more closely than enzymes in cell-free extracts or cell homogenates. Both dehydratases in situ underwent rapid "suicidal" inactivation by glycerol during catalysis, as they do in vitro. The inactivated dehydratases in situ, however, were rapidly and continually reactivated by adenosine 5'-triphosphate (ATP) and Mn2+ in the presence of free adenosylcobalamin, although in cell-free extracts or in cell homogenates they could not be reactivated at all under the same reaction conditions. ATP was partially replaced by cytidine 5'-triphosphate or guanosine 5'-triphosphate but not by the beta, gamma-methylene analog of ATP in the in situ reactivation. Mn2+ was fully replaced by Mg2+ but only partially by Co2+. Hydroxocoblamin could not replace adenosylcobalamin in reactivation mixtures. The ability to reactivate the glycerol-inactivated dehydratases in situ was only seen in cells grown anaerobically in glycerol-containing media. This suggests that some factor(s) required for in situ reactivation is subject to induction by glycerol. Of the two possible mechanisms of in situ reactivation, i.e., the regeneration of adenosylcobalamin by Co-adenosylation of the bound inactivated coenzyme moiety (B12-adenosylation mechanism) and the displacement of the bound inactivated coenzyme moiety by free adenosyl-cobalamin (B12-exchange mechanism), the former seems very unlikely from the experimental results.

Adenosine Triphosphate↗
PubMed1979

Fermentation of 1,2-propanediol with 1,2-ethanediol by some genera of Enterobacteriaceae, involving coenzyme B12-dependent diol dehydratase.

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.

Aerobiosis↗
PubMed1977

Effect of chemical modification of cell surface components of a brewer's yeast on the floc-forming ability.

Effects of treatments with proteolytic enzymes and protein-modifying reagents on flocculation of brewer's yeast IFO 2018 were investigated. The floc-forming ability of the yeast cells was irreversibly eliminated by treatment with papain, trypsin, chymotrypsin or pepsin, indicating that certain proteins on the cell surface participate in the yeast flocculation. Chemical modification with reagents, known to act on disulfide bridges, carboxyl and/or phosphate groups, phenolic groups, amino groups, and imidazole groups, also destroyed the ability to flocculate, although in some cases a high concentration (8 M) of urea was necessary in addition to protein-modifying reagents. Thus, it is suggested strongly that these functional groups of amino acid residues of the proteins are essential for the floc-forming ability of brewer's yeast cells.

Cell Wall↗
PubMed1977

Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase.

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-B12-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-B12-dependent diol dehydratase in a 1,2-propanediol-containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.

Cations, Monovalent↗
PubMed1977

Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme.

A series of 16 analogs of 5'-deoxy-5'-adenosylcobalamin (adenosylcobalamin) were examined for their effects on the diol dehydrase system of Klebsiella pneumoniae (Aerobacter Aerogenes). Four analogs, ara-adenosyl-, aristeromycyl-, 3-isoadenosyl-, and nebularylcobalamin, were able to function as coenzymes in the diol dehydrase reaction, coenzyme activity decreasing in that order. Like the native holoenzyme, complexes of the enzyme with these four analogs show a cob(II)alamin-like absorption peak or shoulder in the presence of 1,2-propanediol. Analogs containing hypoxanthine, cytosine, or benzimidazole do not function as coenzymes, but are weak competitive inhibitors in the presence of adenosylcobalamin. Analogs in which the D-ribosyl moiety is replaced by L-ribose or by an alkyl chain of 2 to 6 carbons are inactive as coenzymes, but act as competitive inhibitors with extremely high affinity for the apoenzyme. Complexes with the inactive analogs showed visible spectra similar to those of the corresponding free cobalamins. Upon anaerobic photolysis and subsequent aeration, complexes with the first group of inactive analogs produced unusually stabilized cob(II)alamin, while complexes with the second group of inactive analogs were readily photolyzed to a hydroxocobalamin-enzyme complex. Complexes with adeninylpentyl- and L-adenosylcobalamin were stable to light under the same conditions. These findings suggest that both the ribose and the adenine moiety of the nucleoside participate in enzyme-coenzyme interaction, involving not only the binding to the apoenzyme but also the activation of the carbon-cobalt bond.

Apoenzymes↗
PubMed1976

Mechanism of inhibition of Chromatium D growth by L-methionine. Regulation of L-threonine biosynthesis by the intracellular level of S-adenosylmethionine.

(1) An unusual accumulation of S-adenosyl-L-methionine in Chromatium D was associated with a marked growth inhibition by L-methionine. The inhibition was overcome by L-isoleucine, L-leucine, L-phyenylalanine, L-threonine, L-valine and putrescien. Based on their effects, these compounds are classified into 3 types. (2) L-Isoleucine, L-leucine, L-phyenylalanine and L-valine (Type I) inhibited the L-methionine uptake and consequently prevented the bacterium from the unusual accumulation of S-adenosyl-L-methionine even in the presence of L-methionine in the medium. Putrescine (Type II) stimulated the consumption of S-adenosyl-L-methionine, but did not influence the L-methionine uptake. Hence, the effect of putrescine would be explained by the action to diminish the intracellular level of S-adenosyl-L-methionine. L-Threonine (Type III) neither inhibited the L-methionine uptake nor affected the content of S-adenoxyl-L-methionine due to the addition of L-methionine. (3) The specific activity of homoserine kinase (EC 2.7.1.39) was greatly lowered by the addition of L-methionine under conditions in which Chromatium D unusually accumulates S-adenoxyl-L-methionine. Homoserine dehydrogenase (EC 1.1.1.3) activity was inhbitied by S-adenosyl-L-methionine (50% inhibition index, 3.5 mM). These facts strongly suggest that the growth inhibition by L-methionine is associated with the L-threonine deficiency caused by the unusual accumulation of S-adenosyl-L-methionine.

Amino Acids↗
PubMed1976

Affinity chromatography of amine oxidase from Aspergillus niger.

Omega-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of epsilon-aminopentyl-Sepharose or delta-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 A) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to omega-aminohexyl-Sepharose. It was also demonstrated that the affinity chromatography on omega-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.

Aspergillus niger↗
PubMed1976

Studies on metabolic role of 5'-Methylthioadenosine in Ochromonas malhamensis and other microorganisms.

Several compound containing a thiomethyl group were found to replace vitamin B12 in a protozoan, Ochromonas malhamensis. The order of the effectiveness was as follows: 5'-methylthioadenosine is greater than S-adenosylmethionine is greater than 5-methylthioribose is greater then L-methionine. A similar order was obtained with respect to the permeability of these compounds into the protozoan cells, except for S-adenosylmethionine. 5'-Methylthioadenosine and 5-methylthioribose as well as L-methionine markedly increased the intracellular content of L-methionine. The level of S-adenosylmethionine was also increased by them, but to lesser degree. The thiomethyl group of the compounds was established to be incorporated into S-adenosylmethionine. The metabolic fate of the thiomethyl group of 5'-methylthioadenosine cannot be distinguished from that of L-methionine. A high activity of 5'-methylthioadenosine nucleosidase was detected in the cell-free extracts of the protozoan. These results strongly suggest that 5'-methylthioadenosine would be metabolized to L-methionine would be ocnverted to S-adenosylmethionine. Like L-methionine and vitamin B12, 5'-methylthioadenosine and 5-methylthioribose may play an important role in maintenance of the C-1 pool in Ochromonas malhamensis. Neither 5'-methylthioadenosine nor 5-methylthioribose replaced vitamin B12 in some vitamin B12-requiring bacteria. This result is consistent with the fact that neither compounds was significantly taken up by these bacteria.

Adenosine↗
PubMed1975

Immobilized diol dehydrase and its use in studies of cobalamin binding and subunit interaction.

Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.

Apoenzymes↗
PubMed1975

Preparation, properties and biological activities of succinyl derivatives of vitamin B12.

Three new derivatives of vitamin B12,0-2'-succinyl-, 0-5'-succinyl-, and 0-2', 0-5'-disuccinyl-vitamin B12, whose alpha-ribose moieties of the nucleotide ligand are succinylated, were prepared by reaction of the vitamin with succinic anhydride. The first succinylation took place rapidly and almost predominantly on 5'-OH of alpha-ribose, and the second succinylation much more slowly on 2'-OH. From the behaviors in paper electrophoresis and the lability to CH- of cobalt-base bond of 0-2'-succinylated vitamin B12 derivatives, it was suggested that the terminal COOH of the 0-2'-succinyl group forms an inner salt with the imidazole nucleus of 5,6-dimethylbenzimidazole. Monosuccinyland disuccinyl-vitamin B12 by mild acid or base hydrolysis. Heating at 130 degrees for 5 min also led to the complete severance of the succinyl group of 0-5'-succinyl-vitamin B12. None of the three succinly derivatives inhibited the diol dehydrase reaction when added with coenzyme B12, SUggesting that the ability to bind to the apoenzyme is strongly diminished or almost lost by succinylation on 2'- or 5'-OH of alpha-ribose. None of the succinyl vitamin B12 compounds showed either biological activity or anti-vitamin B12 activity when tested with Escherichia coli 215, a methionine-B12 auxotroph. None of them significantly inhibited [3-H] vitamin B12 uptake by E. coli 215 cells. This observation implies that succinyl derivatives of vitamin B12 are hardly incorporated into the cells of E. coli.

Biological Transport↗