Steady state kinetics of enzymes with substrates having multiple reaction sites.

An enzyme catalysing the reaction of a substrate with multiple reaction sites may display steady state kinetics described by a Michaels-Menten equation. The Km is identical for all sites considered individually and all sites together. The maximum velocity for a single site depends on the rate constants for reaction at that site and at all of other sites.

Metabolism of 2,6-dimethylnaphthalene by flavobacteria.

Flavobacteria that were able to grow on 2,6-dimethylnaphthalene (2,6-DMN) were isolated from soil. Most were able to oxidize a broad range of aromatic hydrocarbons after growth on 2,6-DMN at rates comparable to that of the oxidation of 2,6-DMN itself. One small group was neither able to grow on naphthalene nor able to oxidize this compound after growth on 2,6-DMN, but metabolized 2,6-DMN by a pathway which converged with that previously described for naphthalene metabolism in pseudomonads. These organisms could also grow on salicylate or methylsalicylate, and in so doing, early enzymes for 2,6-DMN metabolism were induced.

Phthalate pathway of phenanthrene metabolism: formation of 2'-carboxybenzalpyruvate.

The metabolism of phenanthrene by a gram-negative organism able to use this compound as a sole source of carbon and energy has been examined. 1-Hydroxy-2-naphthoic acid was oxidized by oxygen in a reaction catalyzed by a dioxygenase which was activated by ferrous ions. The stoichiometry of the oxidation and the UV spectrum of the product were consistent with the identification of the product as 2'-carboxybenzalpyruvate. This was confirmed by cleaving the product with a partially purified aldolase to yield 2-carboxybenzaldehyde and pyruvate. A number of enzymes for the metabolism of 1-hydroxy-2-naphthoic acid were induced by growth on phthalate or (less well) by growth on protocatechuate. The latter supported only a slow rate of growth, and this and poor induction may have been due to a slow rate of entry into the cell.

Bacterial oxidation of naphthalene and phenanthrene.

An organism grown on both of the hydrocarbons naphthalene and phenanthrene induces a separate enzyme for the initial oxidation of each.

Naphthalene plasmids in pseudomonads.

A rapid method beginning with the direct lysis of bacteria in alkaline sodium dodecyl sulfate was used to detect naphthalene plasmids in pseudomonads. The strains NCIB 9816, PG, ATCC 17483, and ATCC 17484, which can grow on naphthalene as the sole source of carbon and energy, were examined. All except ATCC 17483 contained more than one plasmid. ATCC 17483 did not contain any plasmids. The largest pair of plasmids found in each of NCIB 9816 and PG(NAH2 and NAH3, respectively) determined naphthalene metabolism and could be transferred by conjugation. This also transferred the unusually regulated meta pathway enzymes for catechol metabolism. NAH2 determines the constitutive production of low concentrations of catechol 2,3-dioxygenase and 2-hydroxymuconic acid semialdehyde dehydrogenase, and NAH3 determines the constitutive production of high concentration of these. NAH2 and NAH3 gave identical fragments on digestion with BamHI or HindIII, but these were quite different from those of NAH. Nonetheless, NAH2 and NAH3 hybridized with NAH.

Naphthalene metabolism by pseudomonads: purification and properties of 1,2-dihydroxynaphthalene oxygenase.

1,2-Dihydroxynaphthalene oxygenase was purified from Pseudomonas putida NCIB 9816 grown on naphthalene as the sole source of carbon and energy. The enzyme had a subunit molecular weight of 19,000 and in a medium containing phosphate buffer, 1 mM mercaptoethanol, and 10% (vol/vol) ethanol had a native molecular weight greater than 275,000. The enzyme required Fe2+ for activity. It was inactivated slowly on standing, and inactivation was accelerated by dilution with aerated buffers and by H2O2. Bathophenanthroline sulfonate, o-phenanthroline, 8-hydroxyquinoline, and 2,2'-dipyridyl also inhibited the enzyme. The inactive enzyme was reactivated by anaerobic incubation with ferrous sulfate and ferrous ammonium sulfate. Thiol reagents and acetone, ethanol, or glycerol decreased the rate of loss of activity. The enzyme was most active with 1,2-dihydroxynaphthalene, for which the Km was 2.8 X 10(-4) M. 3-Methyl- and 4-methylcatechols were oxidized at 3 and 1.5%, respectively, of the rate of 1,2-dihydroxynaphthalene, and the Km for 3-methylcatechol was 1.5 X 10(-4) M. Purified 1,2-dihydroxynaphthalene oxygenase catalyzed the oxidation of 1,2-dihydroxynaphthalene, leading to the appearance of 2-hydroxychromene-2-carboxylic acid, but 3-methylcatechol was oxidized by this enzyme to 2-hydroxy-6-oxoheptadienoic acid. Thus, a product structurally analogous to 2-hydroxychromene-2-carboxylic acid was not observed when 3-methylcatechol was oxidized. This may indicate that 2-hydroxychromene-2-carboxylic acid results from cyclization of a ring fission product before release from the enzyme.

Metabolism of naphthalene by pseudomonads: salicylaldehyde as the first possible inducer in the metabolic pathway.

Pseudomonas ATCC 17483 produced enzymes for naphthalene metabolism when growing in a medium containing succinate and naphthalene. Mutants for naphthalene metabolism produced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were able to produce these enzymes only when the metabolic pathway was intact as far as salicylaldehyde, which was therefore identified as the first possible inducer.

The metabolism of S-carboxyalkylcysteines in man.

1. The ingestion of S-(2-carboxy-1-methylethyl)-, S-(2- carboxy-1-methylpropyl)-, S-(1-carboxy-2methylpropyl)- and S-(2-carboxyethyl)-L-cysteine by man was followed by the excretion in the urine of the unchanged amino acid and the N-acetylated compounds. 2. In the rat the fraction of S-(2-carboxy-1-methylethyl)-L-[U-14C]cysteine acetylated increased as the dose level decreased. 3. The significance of these results for the excretion of S-carboxyalkyl-cysteines in man is discussed.

Role and regulation of the ortho and meta pathways of catechol metabolism in pseudomonads metabolizing naphthalene and salicylate.

The enzymes of naphthalene metabolism are induced in Pseudomonas putida ATCC 17484, PpG7, NCIB 9816, and PG and in Pseudomonas sp. ATCC 17483 during growth on naphthalene or salicylate; 2-aminobenzoate is a gratuitous inducer of these enzymes. The meta-pathway enzymes of catechol metabolism are induced in ATCC 17483 and PPG7 during growth on naphthalene or salicylate or during growth in the presence of 2-aminobenzoate, but in ATCC 17484 and NCIB 9816 the ortho-pathway enzymes of catechol metabolism are induced during growth on naphthalene or salicylate. 2-Aminobenzoate does not induce any enzymes of catechol metabolism in the latter two organisms. In Pseudomonas PG the meta-pathway enzymes are present at high levels under all conditions of growth, but this organism and PpG7 can induce ortho-pathway enzymes during naphthalene or salicylate metabolism. Salicylate appears to be the inducer of the enzymes of naphthalene metabolism in all of the organisms studied and, where they are inducible, of the meta-pathway enzymes, but the properties of Pseudomonas PG suggest that separate, regulatory systems may exist.

The bacterial degradation of fluoranthene and benzo[alpyrene.

A gas chromatographic method to determine fluoranthene and benzo[a]pyrene at concentrations close to the limit of their solubility in water was used to identify several species of pseudomonads able to degrade fluoranthene and benzol[a]pyrene. Degradation occurs most rapidly in cultures in the stationary phase, is heat sensitive, requires oxygen, and is enhanced in the presence of cyanide.

The conjugation of glutathione with unsaturated acyl thiol esters and the metabolic formation of S-carboxyalkylcysteines.

The partial purification and properties of an enzyme from the soluble fraction of rat liver that catalyses the reaction of glutathione with 2,3-unsaturated acyl thiol esters is described, and its possible role in the formation of S-carboxyalkylcysteines is discussed. The synthesis of S-(3-methylcrotonyl)- and S-(2-methylcrotonyl)-N-acetylcysteamine and of S-crotonyl-NN-dimethylcysteamine hydrochloride and dicyclohexylammonium S-crotonyl-N-acetyl-l-cysteine is described.

The metabolism of S-methyl-L-cysteine.

1. Methylsulphinylacetic acid, 2-hydroxy-3-methylsulphinylpropionic acid and methylmercapturic acid sulphoxide (N-acetyl-S-methyl-l-cysteine S-oxide) were isolated as their dicyclohexylammonium salts from the urine of rats after they had been dosed with S-methyl-l-cysteine. 2. A fourth sulphoxide was isolated but not identified. 3. The excretion of sulphate in the urine of rats dosed with S-methyl-l-cysteine was measured. 4. The metabolism of S-methyl-l-cysteine by the hamster and guinea pig was examined chromatographically. 5. The preparation of the following compounds is reported: (-)-dicyclohexylammonium methyl-mercapturate sulphoxide; the dicyclohexylammonium salts of the optically inactive forms of 2-hydroxy-3-methylthiopropionic acid, 2-hydroxy-3-methyl-sulphinylpropionic acid and methylsulphinylacetic acid.