Stereospecific microbial reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl-1H-1)-benzazepin+ ++-2-o ne.

A key intermediate, (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluorome thyl)- 2H-1-benzazepin-2-one (compound II or SQ32191), with high optical purity was made by the stereoselective microbial reduction of the parent ketone 1. Several strains of bacterial and yeast cultures were screened for the ability to catalyse the stereoselective reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-1-benzazepin++ +-2,3-dione [compound I or SQ32425]. Microorganisms from the genera Nocardia, Rhodococcus, Alkaligenes, Corynebacterium, Arthrobacter, Hansenula, and Candida reduced compound I to compound II with 60-70% conversion yield. In contrast, microorganisms from the genera Pseudomonas and Acinetobacter reduced compound I stereospecifically to (trans)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromet hyl-2H- 1-benzazepin-2-one (compound III or SQ32408). Among various cultures evaluated, N. salmonicolor SC6310 effectively catalysed the transformation of compound I to compound II with 96% conversion yield at 1.5-2.0 gl-1 concentration. Compound II was isolated and identified by NMR analysis, mass spectrometry, and comparison to an authentic sample. Preparative scale fermentation process and transformation process were developed using cell suspensions of N. salmonicolor SC6310 to catalyse the transformation of compound I to compound II. The isolated compound II had a melting point of 222 degrees C (reference 221-223 degrees C), optical rotation of +130.4 (reference +128 degrees C), and optical purity of greater than 99.9% as analyzed by NMR and chiral HPLC.     

Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2(-)) were investigated. Reduction of compound I by NO2(-) was rapid (k2 = 2.3 x 10(7) M(-1) x s(-1); pH = 7.2) and compound II was not an intermediate, indicating that NO2* radicals are not produced when NO2(-) reacts with compound I. The second-order rate constant for the reaction of compound II with NO2(-) at pH = 7.2 was 3.5 x 10(5) M(-1) x s(-1). The reaction of compound III with NO2(-) exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2(-) concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2(-) complex. The Km of compound III for NO2(-) was 1.7 x 10(-4) M and the first-order decay constant of the compound III/ NO2(-) complex was 12.5 +/- 0.6 s(-1). The second-order rate constant for the reaction of the complex with NO2(-) was 3.3 x 10(3) M(-1) x s(-1). Rate enhancement by NO2(-) does not require NO2* as a redox intermediate. NO2(-) accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2(-) with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Oxidation of clozapine and ascorbate by myeloperoxidase.

The kinetics and spectra of the reactions of clozapine with compounds I and II of myeloperoxidase were investigated using both single- and sequential-mixing stopped-flow techniques, steady-state kinetics, and spectrophotometric measurements. The results show conclusively that both compounds I and II are reduced in one-electron reactions with clozapine. At pH 7.0 the rate constant for compound I reacting with clozapine is (1.5 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (4.8 +/- 0.1) x 10(4) M(-1) s(-1). The physiological pH of 7.4 was found to be optimal for the oxidation of clozapine by compound I. The rate constant for compound I reacting with ascorbate is (1.1 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (1.1 +/- 0.2) x 10(4) M(-1) s(-1), both obtained at pH 7.0. Experiments with both clozapine and ascorbate present showed that ascorbate acts both as a competitive inhibitor and free radical scavenger.     

5-Phenyl substituted 1-methyl-2-pyridones and 4'-substituted biphenyl-4-carboxylic acids. synthesis and evaluation as inhibitors of steroid-5alpha-reductase type 1 and 2.

The synthesis of a series of 5-phenyl substituted 1-methyl-2-pyridones (I) and 4'-substituted biphenyl-4-carboxylic acids (II) as novel A-C ring steroidomimetic inhibitors of 5alpha-reductase (5alphaR) is described. Compounds 1-4 (I) were synthesized by palladium catalyzed cross coupling (Ishikura) reaction between diethyl(3-pyridyl)borane and aryl halides (1b-4b) followed by alpha-oxidation with sodium ferrocyanate of the 1-methyl-pyridinium salt. Inhibitors II (5-18) were obtained either by two successive Friedel-Crafts acylations from biphenyl (5a-10a) followed by saponification to yield the corresponding carboxylic acids (5-10) or by Suzuki cross coupling reaction to give the 4'-substituted biphenyl-4-carbaldehydes 11a-18a. The latter compounds were subjected to a Lindgren oxidation to yield compounds 11-18. The compounds were tested for inhibitory activity toward human and rat 5alphaR1 and 2. The test compounds inhibited 5alphaR, showing a broad range of inhibitory potencies. The best compound in series I was the N-(dicyclohexyl)-4-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl)benzamide 4 exhibiting an IC(50) value for the human type 2 enzyme of 10 microM. In series II, the most active compound toward human type 2 isozyme was the 4'-(dicyclohexyl)acetyl-4-biphenyl carboxylic acid (10; IC(50)=220nM). Both series showed only marginal activity toward the human type 1 isozyme. In conclusion, the biphenyl carboxylic acids (II) are more appropriate for 5alphaR inhibition than the 5-phenyl-1-methyl-2-pyridones (I). Especially the 4'-carbonyl compounds 5-10 represent new lead structures for the development of novel human type 2 inhibitors.     

Spectral and kinetic studies on eosinophil peroxidase compounds I and II and their reaction with ascorbate and tyrosine.

Eosinophil peroxidase, the major granule protein in eosinophils, is the least studied human peroxidase. Here, we have performed spectral and kinetic measurements to study the nature of eosinophil peroxidase intermediates, compounds I and II, and their reduction by the endogenous one-electron donors ascorbate and tyrosine using the sequential-mixing stopped-flow technique. We demonstrate that the peroxidase cycle of eosinophil peroxidase involves a ferryl/porphyrin radical compound I and a ferryl compound II. In the absence of electron donors, compound I is shown to be transformed to a species with a compound II-like spectrum. In the presence of ascorbate or tyrosine compound I is reduced to compound II with a second-order rate constant of (1.0+/-0.2)x10(6) M(-1) s(-1) and (3.5+/-0.2)x10(5) M(-1) s(-1), respectively (pH 7.0, 15 degrees C). Compound II is then reduced by ascorbate and tyrosine to native enzyme with a second-order rate constant of (6.7+/-0.06)x10(3) M(-1) s(-1) and (2.7+/-0.06)x10(4) M(-1) s(-1), respectively. This study revealed that eosinophil peroxidase compounds I and II are able to react with tyrosine and ascorbate via one-electron oxidations and therefore generate monodehydroascorbate and tyrosyl radicals. The relatively fast rates of the compound I reduction demonstrate that these reactions may take place in vivo and are physiologically relevant.     

Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate, an Intermediate of the Phenanthrene-Degradative Pathway of Nocardioides sp. Strain KP7

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in Nocardioides sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name, trans-2′-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by ¹H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.     

Pharmacological properties of new 1,2-benzisothiazolyloxypropanolamines on cardiac and tracheal beta-receptors.

This paper reports the pharmacological assessment of beta-blocking properties of new benzisothiazole and benzisoxazole derivatives, substituted in position 3-, 5- or 7- with the oxypropanolaminic side chain (I-VI), to of which contain the -OCH3 group in position 3- (III, V) in comparison with propranolol. The results, obtained on isoprenaline-induced chronotropic response of rat isolated atria and on isoprenaline-induced relaxation of guinea-pig tracheal strips precontracted by carbachol, suggest that the compounds (I, II, IV, VI), at variance with the methoxy-substituted (III, V), possess a beta 1-blocking activity 4-300 times lower than propranolol. pA2 values drop from 8.36 to 7.56 and 7.04 from the relative compounds substituted in position 7- (IV), 3- (I) and 5- (II), thus indicating that the position of the oxypropanolaminic chain in the benzisothiazole ring affects the ability of the molecules to interact with the beta 1-adrenoceptor. Furthermore, benzisothiazole rather than benzisoxazole ring seems to facilitate the drug-beta 1 adrenoceptor interaction, the compound (I) displaying a 10-fold higher affinity than compound (VI).     

Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide.

The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin pi-cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [lambda(max) (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [lambda(max) (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a 'ferric-like' species (lambda(max) = 406 nm) that is not spectroscopically identical to ferric rAPX (lambda(max) = 403 nm) with a first order rate constant, k(decay)' = (2.7 +/- 0.3) x 10(-4) s(-1). Authentic samples of compound II evolve to ferric rAPX [k(decay) = (1.1 +/- 0.2) x 10(-3) s(-1)]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein-based radical, with g values for compound I* (g parallel = 2.038, g perpendicular = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g parallel = 2.037, g perpendicular = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the 'ferric-like' rAPX followed by RP-HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein-based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.     

Microbiological degradation of bile acids. Metabolites formed from 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl) propionic acid by Streptomyces rubescens.

1. The metabolism of 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl)propionic acid (III), which is a possible precursor of 2,3,4,6,6a beta, 7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-1H-cyclopenta[f]quinoline-3,7-dione (II) formed from cholic acid (I) by streptomyces rubescens, was investigated by using the same organism. 2. This organism effected amide bond formation, reduction of the carbonyl groups, trans alpha beta-desaturation and R-oriented beta-hydroxylation of the propionic acid side chain and skeleton cleavage, and the following metabolites were isolated as these forms or their derivatives: compound (II), 1,2,3,4 a beta,-5,6,6a beta,7,8,9a alpha,9b beta-dodecahydro-6a beta -methylcyclopental[f][1]benzopyran-3,7-dione (IVa), (1R)-1,2,3,4a beta,5,6,6a beta,7,8,9.9a alpha,9b beta-dodecahydro-1-hydroxy-6a beta-methylcyclopenta[f][1]benzopyran-3,7-dione (IVb), (E)-3-(3aalpha-hexahydro-5 alpha-hydroxy-7a beta-methyl-l-oxo-indan-4 alpha-yl)prop-2-enoic acid (V), (+)-(5R)-5-methyl-4-oxo-octane-1,8-dioic acid (VI), 3-(4-hydroxy-5-methyl-2-oxo-2H-pyran-6-yl)propionic acid (VII) and 3-(3a alpha-hexahydro-1 beta-hydroxy-7a beta-methyl-5-oxoindan-4 alpha-yl)propionic acid (VIII). The metabolites (IVb), (V), (VI) and (VII) were new compounds, and their structures were established by chemical synthesis. 3. The question of whether these metabolites are true degradative intermediates is discussed, and a degradative pathway of compound (III) to the possible precursor of compound (VII), 7-carboxy-4-methyl-3,5-dioxoheptanoyl-CoA (IX), is tentatively proposed. The further degradation of compound (IX) to small fragments is also considered.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

The arsonomethyl group as an analogue of phosphate. An X-ray investigation.

The X-ray structure analysis of three compounds of interest as enzyme substrates is reported. They are the hydrated forms of (I) DL-2-amino-4-arsonobutanoic acid [HO-AsO2--CH2-CH2-CH(NH3+)-CO2H], (II) DL-2-amino-4-phosphonobutanoic acid [HO-PO2--CH2-CH2-CH(NH3+)-CO2H] and the hydrated barium salt of (III) D-3-phosphoglycerate [HO-PO2--O-CH2-CH(OH)-CO2-]. The structures were fully refined to R factors of 0.033, 0.053 and 0.046. For the compounds (I) and (II) the charge distribution was directly determined by locating all H atoms. The co-ordination around As and P is approximately tetrahedral, with the valency angle between the two charged O atoms enlarged to 112 degrees in compound (I), 166 degrees in compound (II) and 122 degrees in compound (III). The As-X bond distances are increased relative to P-X to accommodate the increased atomic radius. The analysis establishes that the compounds are structural analogues. Tables of co-ordinates for H atoms, anisotropic thermal parameters, bond lengths and bond angles for the three compounds have been deposited as Supplementary Publication SUP 50122 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained directly [see Biochem J. (1983) 209, 5].     

Arylglycerol-γ-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic β-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-γ-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-β-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a ¹³C-labeled compound (compound II′) indicated that the formyl group of compound IV was derived from the β-phenoxyl group of β-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (compound III). γ-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-β,γ-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with ¹³C-labeled compounds II′ and III′ indicated that the carbonate carbon of compound V was derived from the β-phenoxyl groups of β-O-4 substructure.     

Avidin binding of radiolabeled biotin derivatives

Three N-acyl derivatives of biotinylethylenediamine were prepared: I, biotinylamidoethyl-3-(3-[125I]iodo-4-hydroxyphenyl)propionamide; II, biotinylamidoethyl-[3H]acetamide; and III, biotinylamidoethyl-3-(3,5-[125I]diiodo-4-hydroxyphenyl)propionamid e. Each compound was combined with a large excess of avidin, yielding 1:1 molar complexes. Aside from a small fraction of each complex that dissociated more rapidly, the dissociation half-lives of these complexes were: I, 41 days; II, 4.4 days; and III, 148 days. The iodo- (mono or di) hydroxyphenylpropionyl moieties of I and III, therefore, contribute significantly to the binding strength of these compounds toward avidin. We also formed 4:1 complexes of I, II, and III with avidin (compound in excess), each of which exhibited biphasic dissociation, with initial half-lives of 4, 3.2, and 24 days, respectively. Thus, I or especially III potentially can be used as a sensitive tracer in quantitative studies with avidin.     

The inhibitory effects of phenolic Mannich bases on carbonic anhydrase I and II isoenzymes

Phenolic mono Mannich bases [2-[4-hydroxy-3-(aminomethyl)benzylidene]-2,3-dihydro-1H-inden-1-one (8–15)] and bis Mannich bases [2-[4-hydroxy-3,5-bis(aminomethyl)benzylidene]-2, 3-dihydro-1H-inden-1-one (2–7)] were synthesized starting from 2-(4-hydroxybenzylidene)-2, 3-dihydro-inden-1-one (1). This study was designed in order to investigate the carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties of a library of compounds incorporating the phenol functional group. All prepared compounds showed a low inhibition percentages on both human (h) isoforms hCA I and hCA II compared to the reference sulfonamide acetazolamide. Mannich bases 2–15 had lower inhibition percentages than the compound 1 on hCA I and hCA II, except compound 14, which is a Mannich base derivative of dipropylamine, which had a similar inhibitory power as compound 1 on hCA II. All compounds synthesized 1–15 were 1.3–1.9 times more effective on hCA II comparing with the effectivenes of the compounds on hCA I.     

Arylglycerol-gamma-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic beta-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-gamma-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-beta-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-beta-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a C-labeled compound (compound II') indicated that the formyl group of compound IV was derived from the beta-phenoxyl group of beta-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether (compound III). gamma-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-beta,gamma-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with C-labeled compounds II' and III' indicated that the carbonate carbon of compound V was derived from the beta-phenoxyl groups of beta-O-4 substructure.     

The identification and biosynthesis of siderochromes formed by Micrococcus denitrificans.

1. Micrococcus denitrificans excretes three catechol-containing compounds, which can bind iron, when grown aerobically and anaerobically in media deficient in iron, and anaerobically in medium with a high concentration of Ca2+. 2. One of these compounds was identified as 2,3-dihydroxybenzoic acid (compound I), and the other two were tentatively identified as N1N8-bis-(2,3-dihydroxybenzoyl)spermidine (compound II) and 2-hydroxybenzoyl-N-L-threonyl-N4[N1N8-bis-(2,3-dihydroxybenzoyl)]spermidine (compound III). 3. The equimolar ferric complex of compound III was prepared; compound III also forms complexes with Al3+, Cr3+ and Co2+ ions. 4. Cell-free extracts from iron-deficient organisms catalyse the formation of compound II from 2,3-dihydroxybenzoic acid and spermidine, and of compound III from compound II, L-threonine and 2-hydroxybenzoic acid; both reactions require ATP and dithiothreitol, and Mg2+ stimulates activity. The enzyme system catalysing the formation of compound II has optimum activity at pH 8.8 Fe2+ (35muM), Fe3+ (35muM) and Al3+ (65muM) inhibit the reaction by 50 percent. The enzyme system forming compound III has optimum activity at pH 8.6. Fe2+ (110 muM), Fe3+ (110 muM) and Al3+ (135 muM) inhibit the reaction by 50 percent. 5. At least two proteins are required for the formation of compound II, and another two proteins for its conversion into compound III. 6. The changes in the activities of these two systems were followed after cultures became deficient in iron. 7. Ferrous 1,10-phenanthroline is formed when a cell-free extract from iron-deficient cells is incubated with the ferric complex of compound III, succinate, NADH and 1,10-phenanthroline under N2.     

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.     

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.     

Microbiological degradation of bile acids. The preparation of some hypothetical metabolites involved in cholic acid degradation.

1. To identify the intermediates involved in the degradation of cholic acid, the further degradation of (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IVa) by Arthrobacter simplex was attempted. The organism could not utilize this acid but some hypothetical intermediate metabolities of compound (IVa) were prepared for later use as reference compounds. 2. The nor homologue (IIIa) and the dinor homologue (IIIb) of compound (IVa) were prepared by exposure of 3-oxo-24-nor-5beta-cholan-23-oic acid (I) and (20S)-3beta-hydroxy-5-pregnene-20-carboxylic acid (II) to A. simplex respectively. These compounds correspond to the respective metabolites produced by the shortening of the valeric acid side chain of compound (IVa) in a manner analogous to the conventional fatty acid alpha- and beta-oxidation mechanisms. Their structures were confirmed by partial synthesis. 3. The following authentic samples of reduction products of the oxodicarboxylic acids (IIIa), (IIIb) and (IVa) were also synthesized as hypothetical metabolities: (4R)-4-[3aalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]valeric acid (Vb) and its nor homologue (VIIa) and dinor homologue (IXa);(4R)-4-[3Aaalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]-pentan-1-ol (Vc); and their respective 5beta epimers (Ve), (VIIc), (IXc) and (Vf). 4. In connexion with the non-utilization of compound (IVa) by A. simplex, the possibility that not all the metabolites formed from cholic acid by a certain micro-organism can be utilized by the same organism is considered.